Heading toward Macromolecular and Nanosized Bioresponsive MRI

Article pubs.acs.org/accounts

Heading toward Macromolecular and Nanosized Bioresponsive MRI Probes for Successful Functional Imaging Goran Angelovski* MR Neuroimaging Agents, Max Planck Institute for Biological Cybernetics, D-72076 Tuebingen, Germany CONSPECTUS: The quest for bioresponsive or smart contrast agents (SCAs) in molecular imaging, in particular magnetic resonance imaging (MRI), is progressively increasing since they allow for the monitoring of essential biological processes on molecular and cellular levels in a dynamic fashion. These are offshoot molecules of common contrast agents that are sensitive to biochemical changes in their environment, capable of reporting on such changes by inducing MRI signal alteration. Various mechanistic approaches and different types of SCAs have been developed in order to visualize desired processes, using diverse imaging protocols and methods. To date, the most frequently exploited probes are paramagnetic molecules that change longitudinal or transverse relaxation at proton frequency, or so-called T1- and T2-weighted probes, respectively. Moreover, SCAs operating by the chemical exchange saturation transfer mechanism, suitable for 19F MRI or possessing hyperpolarized nuclei have also appeared in the past decade, slowly finding their role in functional imaging studies. Following these mechanistic principles, a large number of SCAs suitable for diverse targets have been reported to date. This Account condenses this exciting progress, particularly focusing on probes designed for abundant targets that are suitable for practical, in vivo utilization. To date, the greatest advancements have been certainly made in the preparation of pH sensitive probes, which usually contain protonable groups that interact with paramagnetic centers, or take advantage of supramolecular (dis)assembling to induce the MRI signal change, thereupon enabling pH mapping in vivo. In a complementary approach, a combination of metal chelating ligands for Ca2+ or Zn2+ with MR reporting units results in a wide variety of SCAs that operate with different contrast mechanisms and can be used for initial functional experiments. Finally, the first examples of molecular sensing by creating host−guest complexes to track neurotransmitter flux have also been recently reported, allowing the study of brain function in an unprecedented manner. Nevertheless, wider SCA utilization in vivo has not yet been achieved. There are a few reasons for this disparity between their nominal potential and practical usage, with one of the major reasons being the low sensitivity of the MRI technique. Subsequently, the production of detectable signal change can be achieved using higher concentrations of the bioresponsive probe; however, the biocompatibility of these probes then starts to play an important role. An elegant solution to these practical challenges has been found with the integration of multiple small-sized SCAs into macromolecular and nanosized probes. In such case, the multivalent SCAs are able to circumvent the sensitivity issue, thus enhancing the MR signal and desired contrast changes. Moreover, they prolong the probe tissue retention time, while often reducing their toxicity. Finally, with altered size and properties, they allow for exploitation of mechanisms that induce the contrast change which is not possible with small-sized SCAs. To this end, this Account also discusses the current approaches that aim to develop macromolecular and nanosized SCAs suitable for practical MRI applications. With these, further progress of this exciting field is affirmed, with remarkable results expected in the near future on both the probe preparation and their utilization in functional molecular imaging.

1. INTRODUCTION Molecular imaging aims to characterize and monitor biological processes at the cellular and molecular levels, allowing visualization of organ, tissue, or cellular function without perturbing living organisms. Unlike in common diagnostic imaging, its main goal is to probe and understand the origin and principles of ongoing physiological and pathological processes, rather than to observe their final effects. Among a number of rapidly growing imaging techniques, MRI is one of the best suitable for this purpose: it is noninvasive, has unlimited tissue penetration depth, and excellent spatiotemporal resolution. Although it may exist as a stand-alone technique by using the © 2017 American Chemical Society

intrinsic signal from tissue, the specificity of MRI studies can be dramatically enhanced using various types of CAs which operate at different frequencies or produce MR contrast (i.e., the relative difference in signal intensities between two MR images) using different signal mechanisms.1−3 Moreover, a special class of these probes commonly called (bio)responsive or smart CAs (SCAs) is perfectly suitable for utilization in functional imaging experiments.4,5 Namely, SCAs are capable of varying their MR properties depending on the changes in the Received: April 25, 2017 Published: August 25, 2017 2215

DOI: 10.1021/acs.accounts.7b00203 Acc. Chem. Res. 2017, 50, 2215−2224

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Accounts of Chemical Research

Figure 1. Major mechanisms used in the design of SCAs: (A,B) T1-weighted; (C) T2-weighted; (D) paraCEST, and (E) SCAs suitable for 19F MRI.

common mechanisms that underlie the achieved MR contrast alterations, in specific T1, T2, paraCEST, fluorinated, or hyperpolarized SCAs (Figure 1). CAs for T1-weighted MRI are mainly gadolinium- or manganese-based paramagnetic complexes.1,2 In 1H MRI, the efficacy of a T1-weighted CA to induce the relaxation enhancement of water protons is normalized per millimolar concentration of paramagnetic ion, and is defined as longitudinal relaxivity, r1. SCAs suitable for T1-weighted MRI are capable of altering their r1 along with specific changes in their microenvironment. In order to obtain this effect, at least one of the parameters that determine r1 must be changed, in particular: (a) number of the directly coordinated water molecules in the inner-coordination sphere (q number); (b) the water exchange rate between the inner-sphere and bulk water molecules (kex); (c) rotational correlation time of the complex (τR); (d) the electron spin relaxation times (T1e and T2e).1 Formerly, when most MRI scanners operated at fields < 1.5 T, the mechanism which included change in τR was dominant; nevertheless, with the availability of high- and ultrahigh field scanners (4.7−14.1 T), it is more sensible to develop SCAs that change q or kex. Nowadays, SCAs that undergo q change are the most abundant among the T1-suitable SCAs (Figure 1A,B). T2-Weighted CAs are mainly based on the iron oxide particles, SPIOs and USPIOs, although oxides of paramagnetic metals such as dysprosium or holmium can also be used.7 In analogy to r1, the effect of the CA on T2 is expressed with the term transverse relaxivity, r2. Here, the r2 value is rather

microenvironment which come due to biological alterations in tissue, thus reporting on functional changes in the investigated living systems. Nevertheless, the low inherent sensitivity of MRI forces the evolution of SCAs in the direction of multimeric probe development,6 which can amplify the MR signal and should be capable of producing detectable signal changes in functional studies. Furthermore, the necessity to direct the agent into the target tissue, to improve its biokinetic properties, or to make it suitable for additional imaging readouts is slowly orienting the field in the direction of multifunctional SCAs, i.e., macromolecular and nanosized probes. The purpose of this Account is to briefly summarize the latest developments in this field and indicate approaches for SCA development that can be realistically used for functional MRI, especially pointing out the direction of nanosized SCAs due to their greater potential for practical use.

2. OPERATING CONTRAST MECHANISMS FOR SCAs To date numerous SCAs sensitive to pH, metal ions, different molecules, or enzymes have been reported.1,4,5 Irrespective of the target (i.e., final desired application), these molecules can be chemically classified as organic molecules, chelates of paramagnetic lanthanide or transition metals with different organic ligands, or supramolecular assemblies. In either case, they must follow the fundamental physics principles for generation of the MRI signal and be able to change their specific properties in order to generate stimuli-triggered MRI contrast change. The following section briefly lists the most 2216


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Figure 2. Biotinylated pH reponsive MRI agent with protonable phosphonate group that targets avidin.25

Figure 3. (A) Bismacrocyclic SCA responsive to Ca2+; (B) SCA addition lowers the [Ca2+]i in cultured astrocytes irrespective of the applied concentration; (C) relative declines in [Ca2+]i upon addition of benz, Thg, LaCl3, GdCl3, or 2-APB show existence of Ca2+ transport through the plasma membrane in the presence of SCA (1.2 mM); (D) response of astrocytes to ATP-induced stimulation (1 mM) in the presence of SCA (1.2 mM).34 Reproduced with permission from ref 34. Copyright 2014 Americal Chemical Society.

coating procedures for stabilizing and vectorization purposes,1 various modalities for their clustering or disassembling can be envisaged (Figure 1C).8 The CEST mechanism is, similarly to principles described above, mainly used in 1H MRI,3 albeit approaches for 19F MRI have been also reported.9 This methodology relies on the

influenced by the particle size that substantially affects the diffusion of water molecules on the particle surface. Furthermore, the r2 effect is proportional to the magnetic field, and gets stronger with increasing field. Thus, modulation of the T2-weighted signal is almost exclusively achieved by changing the SCA size. Since the particles undergo various 2217


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Figure 4. Fluorinated SCA suitable for Ca-sensing at two different frequencies (A); its 1H and 19F NMR signal changes (B), and the mechanism which causes q/r1 changes (C).35 Adapted with permission from ref 35. Copyright 2015 Wiley-WCH Verlag GmbH&Co. KGaA, Weinheim.

the metabolism and endogenous ions,14,15 or sensing a broad spectrum of biological targets with the hyperCEST methodology.16

existence of exchangeable protons in endogenous or exogenous CAs which are in slow-to-intermediate exchange with bulk water. The application of a RF pulse matching the frequency of the exchangeable protons leads to magnetization transfer to bulk water, decreasing its MRI signal intensity. Such exchangeable protons are characteristic for several functional groups (amides, amines, hydroxy, inner-sphere coordinated water) with their exchange rates being highly sensitive to environmental changes (Figure 1D). In addition to having different resonant frequencies, these can also be shifted with paramagnetic lanthanide or transition metal ions,1,10 thus avoiding interferences from existing diamagnetic CEST processes. Consequently, a large number of paraCEST SCAs activated by a range of biochemical stimuli have been reported to date.1,3,5 Fluorine-containing molecules are gaining increasing interest due to a high natural abundance and the gyromagnetic ratio of 19 F, while the absence of endogenous fluorine and hence background signal in living systems allow quantitative studies.11 Moreover, minimal changes in the fluorine microenvironment can cause significant changes in the 19F NMR/MRI signal frequency and/or intensity. The 19F signal can also be manipulated analogously to 1H: the use of paramagnetic ions can cause the paramagnetic relaxation enhancement (Figure 1E), while assembling or disassembling of clusters with fluorine atoms can dramatically affect 19F T2 (see above, T2-weighted SCAs). Indeed, due to the lower sensitivity of this methodology, the latter two principles are the most commonly used for the preparation of SCAs suitable for 19F MRI.11,12 Finally, low sensitivity and isotope abundance of several NMR-active nuclei can be successfully circumvented by hyperpolarization. Here, several available techniques alter the nuclear spin population to a level different from the equilibrium defined by the Boltzmann equation, resulting in NMR signal enhancement of several orders of magnitude.2,13 Principally, there is no universal method to induce the signal changes in hyperpolarized SCAs, as it rather depends on the nucleus in question. Nevertheless, detecting the frequency shift difference appears to be the most frequently applied strategy for imaging

3. TYPES OF SCAs FOR PRACTICAL BIOMEDICAL APPLICATIONS The following section summarizes the latest achievements in this field, focusing on SCAs for several biochemical targets

Figure 5. Ca-responsive, paraCEST CAs (Eu- or Yb-based) that change the kex of inner-sphere water or amide protons, respectively, upon Ca2+ binding to imidoacetate arms (blue dashed ellipse).37

which are the most attractive or have already shown the greatest advancements toward final in vivo utilization in functional MRI. Within these, special emphasis is given to emerging approaches with macromolecular and nanosized SCAs. 2218


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complexes with poly-β-CDs that allow quantification of Gd3+ and subsequently pH mapping (see above).27 Alternative approaches to T1-weighted SCAs have been also used for pH sensing. Taking advantage of a substantial difference in r1 and r2 values in the paramagnetic core−shell nanoparticle, their ratio was followed as a function of pH.28 This time the paramagnetic chelate did not undergo any major changes to affect the r1, but the observed changes can be entirely explained with r2 alterations due to shrinking and swelling of the nanoparticle. Analogously, entirely different nanostructures consisting of fluorine-containing micelles were used to provide binary on/off pH transitions by means of 19F MRI.12 Here the higher pH allows for assembling of the micelles which results in the vanishing of the 19F signal due to restricted chain motions. Inversely, micelles disassemble at low pH giving rise to a strong 19F signal. pH sensing has been probed also with paraCEST agents. In fact, since CEST effects heavily depend on water exchange rates, as well as the latter with pH, it is obvious that paraCEST agents are inherently pH sensitive.1 However, due to the lower sensitivity of paraCEST vs T1- or T2-weighted SCAs, attempts to attach a tetraamide complex of Yb3+ to G1 and G3 dendrimers have been also made, showing changes in the CEST effect in the most relevant pH range 6.5−7.5.29 Finally, the first attempts to assess pH with hyperpolarized SCAs have also been made.14 These were small molecules that rapidly penetrate to the desired tissue; the approaches involving macromolecular agents for more sophisticated studies are yet to be developed and reported.

Figure 6. Dendrimeric G1 SCA (A) that remains active in the presence of Ca2+ (B) and exhibits slower difussion in vivo compared to its monomeric SCA analogue (C,D).41 Adapted with permission from ref 41. Copyright 2015 Royal Society of Chemistry.

3.1. pH Sensing

3.2. Metal Ion Sensing

The ability to measure local pH in living organisms is of great biomedical importance. Several imaging techniques, especially MRI, are ideally suitable for pH determinations in vivo.17 Therefore, different approaches using SCAs have been developed, aiming to enable pH mapping. One of the first reported pH-sensitive SCAs was the tetraamide Gd3+ complex, appended with methylenephosphonate groups.18 This complex exhibited a major r1 change in the pH range 6.3−9.5 as a consequence of pH-dependent prototropic exchange of bound water protons influenced by phosphonates. Due to such advantageous properties, this system was used for extracellular pH imaging of rat glioma,19 while its coupling to a PET tracer allowed quantification of the Gd3+ concentration, which enabled r1 determination and the generation of pH maps.20 In parallel, an analogous dendrimerbased SCA was prepared, aiming to increase the r1 response to pH changes.21 Recently, this and analogous bimodal probes were used to demonstrate a concentration-independent method to generate pH maps, or to internalize the sensor into cells in order to measure intracellular pH.22,23 We also used phosphonates as pH-sensitive groups to induce r1 changes in SCAs. A pair of phosphonate-containing Gd3+ complexes exhibited coordination to the lanthanide metal center at higher pH, followed by an increase in q and thus r1 during the protonation of the phosphonate at lower pH.24 Upon synthetic modification of these SCAs and their biotinylation, the pH-dependent relaxometric response remained, as well as in their macromolecular complexes with avidin (Figure 2).25 Similarly, arylsulfonamide-containing SCAs exhibited pHsensitivity.26 Moreover, this group was used to perform further synthetic transformations which resulted in the incorporation of adamantane moieties to form fluorinated supramolecular

Several metals have an essential biological role through involvement in a number of physiological processes. Nevertheless, their monitoring with SCAs is conceivable mainly for metals available in greater than micromolar concentrations due to MRI sensitivity. Calcium and zinc are abundant and the most attractive examples due to their sufficient concentrations and irreplaceable role in neuronal signaling, muscle contraction, or enzymatic reactions. Although essential for life, sodium and potassium are extremely hard to follow by means of functional MRI. Finally, magnesium fulfills most of the requirements, albeit its concentration remains constant during neuronal activity; hence, minor work has been done on its SCAs. Calcium is a favored target due to its robust intra- and extracellular concentrations changes during brain activity, consequently stimulating significant progress in various chemistry disciplines, particularly in the field of Ca-sensitive fluorescent molecular sensors.30 Pioneering steps toward CaMRI sensors were made in the late 1990s by combining a wellknown BAPTA chelator with two DO3A units.31 Despite a Catriggered r1 increase of almost 80% at 11.7 T, the dissociation constant of 0.96 μM made this SCA suitable for intracellular Ca-recordings. This is very hard to achieve, given the low sensitivity of MRI and necessity to deliver the probe inside the cell. Similarly, SPIO particles coated with the protein calmodulin exhibited Ca-induced aggregation and a change in the T2-weighted MRI signal, showing suitability for following submicromolar Ca-levels (0.1−1.0 μM).32 We have performed systematic research implementing the former approach with paramagnetic Gd-based probes. We aimed for targeting extracellular Ca2+ ([Ca2+]e ∼ 1.0−1.4 mM in brain), as its concentration range is comfortable for MRI signal detection and SCA delivery is mitigated (cell internal2219


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Figure 7. Ultrasmall nanoparticle-based SCA interacts with Ca2+ and leads to MRI signal increase in two kidney compartments in vivo.42 Upper row: complexation of SCA with Ca2+ (left) and the relaxometric titration with Ca2+ at 7T (right). Middle row: coronal MR images recorded (a) before and (b) 5 min, (c) 16 min, (d) 37 min, (e) 69 min, and (f) 88 min after the addition of SCA. CaCl2 was added after 11 min (between images b and c). Lower row: plots showing MR signal enhancement of renal pelvis (left) and renal parenhyma (right) of the mice kidneys upon addition of CaCl2 to SCA in vivo. Adapted with permission from ref 42. Copyright 2015 Wiley-WCH Verlag GmbH&Co. KGaA, Weinheim.

This prompted us to investigate further the lead SCA core structure and understand the operating mechanism. We first replaced one macrocycle for a fluorine-containing aryl moiety, showing the potential of Ca-sensitive SCAs suitable for 19F MRI (Figure 4). Equally importantly, having the ability to perform high-resolution and multinuclear NMR studies on these SCAs, we were able to fully understand the mechanism of MR signal change. It involves the hydration change only in the major SAP geometrical isomer, while the minor TSAP isomer appears to be monohydrated already in the Ca-free condition; hence, the latter does not contribute to the overall r1 change.35 Following these mechanistic insights, we attempted to develop a responsive paraCEST probe. Interestingly, replacement of carboxylates for amides in the DO3A chelating unit changed the SAP:TSAP isomer ratio toward the major TSAP species with much faster kex, and hence, no CEST effect was observed at all.36 Nevertheless, we prepared and investigated the first example of a Ca-sensitive paraCEST SCA, which is based on a DOTA-tetraamide chelator by altering the kex through affecting the protonation state of the neighboring imidoacetate side arms (Figure 5).37

ization is not required), albeit the probe affinity had to be adjusted to higher μM − mM levels. Hence, we explored a range of well-known Ca-chelators coupled to one or two MRreporting moieties (Gd-DO3A units), modalities of their attachment to the MR reporter and hence the signal transduction upon Ca2+ binding.33 To date, the best results have been observed with an EGTA-derived chelator, known to be very selective for Ca2+ versus Mg2+, its main competitor in extracellular space. A bismacrocyclic SCA exhibited a Cainduced increase in r1 of over 80% (per Gd3+) in buffered medium, practically resulting in double signal amplification relative to common monomacrocyclic complexes due to the interaction of two Gd3+ ions with a single Ca2+. Our subsequent studies in medium that resembles brain extracellular matrix, complex 3D cell culture model, and conventional 2D cultures of astrocytes showed the excellent potential of this compound class (Figure 3).34 Moreover, the estimates for an idealized in vivo fMRI experiment revealed up to 6% possible change in T1 during intense neuronal activity, indicating this methodology might become superior to BOLD in many neuroimaging investigations since the latter reaches maximal changes on the level of a few percent. 2220


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Figure 10. TACN-containing SCA responsive to Zn2+. The DFT structures represent optimized geometries of the SAP isomers in absence and presence of Zn2+.45 Reproduced with permission from ref 45. Copyright 2015 Americal Chemical Society. Figure 8. Liposome-based SCA that dramatically increases r1 at low magnetic fields (0.5 T) in the presence of Ca2+ (A); the monomeric SCA is not able to induce the same magnitude of r1 increase (B) due to slower rotation of MRI reporting unit in liposome after binding to Ca2+ (C).43 Reproduced with permission from ref 43. Copyright 2015 Americal Chemical Society.

diffusion properties on the potential success of fMRI experiments.38,39 We anticipated that making a nanosized probe with biocompatible nanocarriers will assume the carrier’s pharmacokinetic properties, a high load of the monomeric SCA units will amplify the local MR signal, while the combination of multifunctional carriers with targeting vectors or probes may allow a multimodal readout.40 We therefore incorporated the aryl-isothiocyanate group, or the lipophilic derivative of dioctadecylamine into the SCA chelator. The former amine-reactive SCAs were used for preparation of different generations of PAMAM dendrimers or polysiloxane-based nanoparticles, whereas the liposomic SCAs were prepared with the latter amphiphilic monomacrocycle.41−44 A G1 dendrimeric SCA kept a high relaxivity response to Ca2+ and exhibited much slower diffusion than the monomeric analogue in rat cortex in vivo, proving that incorporation of SCAs into the biocompatible nanosized carriers can result in systems with very advantageous properties (Figure 6).41 More importantly, when the monomeric SCAs were grafted onto nanoparticles known to have an efficient renal clearance, the in vivo studies with this probe showed the MR signal change upon administration of Ca2+ (Figure 7).42 To date, these are the first probes that detect Ca2+ concentration changes by means of MRI in vivo. Using the amphiphilic SCA, we studied liposome-based SCAs that displayed an extraordinary increase in r1 of up to 400% at 0.5 T (Figure 8). This is due to the large probe size (hence slow τR, see above) and the additional local rigidification of the monomeric units upon interaction with Ca2+.43 This probe is open to additional functional improvements, since it allows the incorporation of other markers (e.g., fluoresecence dyes) or targeting vectors in the lipid bilayer, while its innersphere can be filled with other water-soluble functional species. Recently, we also demonstrated a novel approach with nanosized SCAs which may be very beneficial for fMRI approaches. Here, the r1 and r2 responses of higher generation dendrimeric SCA (G4) were investigated as a function of Ca2+

Figure 9. Dendrimeric (G4) SCA that swells and shrinks in the absence and presence of Ca2+, respectively. SCA diameter change affects the r1/r2 ratio, allowing fast gain in MRI signal with bSSFP sequence.44

Our latest attempts were focused on preservation and maximal exploitation of the triggering mechanism that robustly works in Gd-DO3A systems (Figure 1A,B), and using it to prepare multimeric and nanosized probes. Namely, our preliminary MRI studies in vivo with first-generation monomeric SCAs indicated the critical importance of probe’s 2221


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Figure 11. SCAs responsive to amino acid NTs and their response to selected NTs.52,53

concentration. Interestingly, in parallel to expected changes in r1, the changes in r2 were found to be much more pronounced, mainly due to the probe’s shrinking and swelling in the absence and presence of Ca2+, respectively (Figure 9).44 In turn, this change in r1/r2 ratio can be used for rapid image acquisition by means of the bSSFP pulse sequence. The calculated contrast-tonoise gain normalized per image acquisition time was at least three times higher when using G4-SCA with bSSFP compared to any standard imaging sequence used with SCAs. Our future steps include further investigation of this phenomenon and its optimization for use in monitoring of calcium dynamics or other biological processes with high temporal resolution. In addition to calcium, zinc has an important physiological role in enzymatic reactions or neuronal transmission. Although its concentrations in serum are in the low micromolar range, the local abundance in the brain, prostate, or pancreas reaches levels suitable for monitoring its changes by means of MRI. Therefore, zinc as an SCA target was investigated in several studies, involving complexes with various Zn-sensitive chelators. We recently investigated an SCA which consisted of the same MR-reporting moiety as in the Ca-sensitive SCAs, and a TACN macrocycle as a Zn-sensor (Figure 10).45 The addition of Zn2+ resulted in a substantial increase in r1 of over 150%, with excellent selectivity over other endogenous ions, such as Ca2+ or Mg2+. However, the greatest amount of work has been performed with molecules that contain a BPEN unit to result in numerous T1-weighted and paraCEST SCAs.46,47 The most recent studies report on the interaction of the Znsensitive SCA with HSA that results in the formation of an extremely potent nanoassembly, yet used to monitor Zn2+ release in the prostate by means of MRI in vivo.46,48 Namely, while the addition of Zn2+ to SCA in a ratio of 2:1 just moderately increases its r1, the Zn2-SCA complex tends to form a ternary complex with HSA which leads to substantial r1 increase (from 5.6 to 9.4 mM−1 s−1 for SCA and Zn2-SCA-HSA ternary complex, respectively). This signal amplification was used to follow glucose-induced Zn2+ release in healthy versus malignant prostate in vivo, showing that functional MRI can be a valuable asset to current diagnostic approaches that lack the sufficient method specificity.

NTs are additional molecular targets that are extremely attractive for neuroimaging and suitable for fMRI investigations. In fact, a couple of distinct approaches for the development of NT-sensitive SCAs have already been established and they were very recently explored in our review article.51 Here a brief description of these approaches will be given. The former includes genetically encoded proteins with a heme-bound paramagnetic iron in the active site. The resulting product is a high molecular weight SCA, and its nanosize dimensions serve a very specific recognition with the target NT (dopamine or serotonin) which replaces the iron-coordinated water molecule and thus reduces the r1. We have assumed an approach which included using artificial receptors for amino acid NTs.52,53 These SCAs comprise a positively charged Gd-chelating moiety and a crown ether to accommodate zwitterionic NTs. The interaction of the NT with the SCA leads to ternary complex formation which, analogously to the previously mentioned protein-SCA approach, reduces the r 1 (Figures 1B and 11). We demonstrated the great potential of this approach by applying the NT-sensitive SCA on acute brain slices and monitoring the KCl-induced neuronal depolarization using MRI.52 We are currently pursuing this research direction by developing second-generation SCAs that have improved affinity toward the amino acid NTs and possess additional functional groups that make them suitable for coupling to various biocompatible nanocarriers.

4. CONCLUSIONS The current decade has brought us significant advancements in the field of bioresponsive MRI CAs. Beside the development of new SCA classes sensitive to various targets and implementing different mechanisms to generate the MR signal, several responsive probes that are active in vivo were already reported. The current trends head toward preparation and utilization of nanosized SCAs, especially multimeric probes assembled on nanocarriers. The most important reasons for this are signal amplification in the target tissue, improvement in the probe’s biocompatibility and tissue retention time, and also the possibility to functionalize the probe in a straightforward manner in order to expand its scope of application.54 As such, coupling of various functional molecules to nanosized SCAs can allow for a multimodal readout, improve its targeting abilities to accumulate in desired tissue, or help probe quantification, an important issue that is specific for SCAs and their use in fMRI.55 However, the most recent progress in SCA characterization and utilization in vivo has exhibited the paramount prospects of fMRI with these molecular probes. Their forthcoming improvement as well as the expansion and diversification of their use will allow this field to become an

3.3. Sensing of Molecules

Enzymes represent one of the most preferred targets for functional MRI. The SCA field indeed started with pioneering work on a Gd-based probe sensitive to β-galactosidase activity,49 which was subsequently used to visualize gene expression in vivo.50 Following this example, diverse MRI methods and targets have been used to develop enzymesensitive SCAs, using all available mechanisms to generate and follow MRI signal changes. This is a very broad field with many SCA examples, nicely summarized in a recent review article.5 2222


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indispensable tool for noninvasive, molecular-level monitoring and visualization of diverse biological processes.

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AUTHOR INFORMATION

Corresponding Author

*Email: [email protected]. ORCID

Goran Angelovski: 0000-0002-8883-2631 Notes

The author declares no competing financial interest. Biography Goran Angelovski received a diploma degree from the University in Belgrade, Ph.D. degree from the University in Dortmund and venia legendi (habilitation) from the University of Tuebingen. He is currently an independent group leader at the MPI for Biological Cybernetics in Tuebingen. His research interests include the development of responsive molecular probes for functional MRI applications, from their preparation, to the final in vivo utilization.

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ACKNOWLEDGMENTS I thank all collaborators from joint publications for collegial and exciting work, and my current group members for helpful discussions. The financial support of Max Planck Society is gratefully acknowledged.

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ABBREVIATIONS 2-APB, 2-aminoethoxydiphenyl borate; BAPTA, 1,2-bis(oaminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; benz, benzamil hydrochloride; BOLD, blood oxygen level dependent; BPEN, N,N-bis(2-pyridyl-methyl) ethylene diamine; bSSFP, balanced steady-state free precession; CEST, chemical exchange saturation transfer; DFT, density functional theory; DO3A, 1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylic acid; DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate; EGTA, glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; G, generation of the dendrimer; HSA, human serum albumin; MRI, magnetic resonance imaging; NT, neurotransmitter; PET, positron emission tomography; RF, radiofrequency; SAP, square antiprismatic; (S)CAs, (smart) contrast agents; SNR, signal-to-noise ratio; TACN, 1,4,7-triazacyclononane; Thg, thapsigargin; TSAP, twisted-square antiprismatic; (U)SPIO, (ultra)small particles of iron oxide

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REFERENCES

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Heading toward Macromolecular and Nanosized Bioresponsive MRI

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