Supramolecular design based activatable magnetic resonance imaging

Magnetic resonance imaging (MRI) has been widely used in clinical diagnosis. In recent years, activatable MRI probes responding to specific changes in their microenvironment have been developed. The signal of the MRI probe can be modulated based on supramolecular design, which can include properties such as self‐assembly and molecular recognition. In this review, we summarize the activatable MRI probes based on the design principles of supramolecular chemistry. We also discuss the current challenges and the future perspectives for how activatable MRI can achieve more extensive clinical applications for cancer therapy.


INTRODUCTION
Magnetic resonance imaging (MRI), as an imaging technique based on the phenomena of nuclear magnetic resonance, 1 has been widely used in clinical diagnosis because of its unlimited depth of tissue penetration, noninvasive imaging, high spatial resolution, and tomographic capability. 2 In a strong external magnetic field, magnetic atomic nuclei can absorb radio-frequency pulses with specific wavelengths. In a typical MRI experiment, a radio-frequency pulse causes the magnetization vector of some magnetic nuclei change from parallel to the external field to transverse to the external field. Nuclear relaxation to the equilibrium state can occur through T 1 (longitudinal) and T 2 (transverse) mechanisms. Images are usually generated by monitoring nuclear relaxation after radio-frequency pulses. 3 To overcome the low sensitivity inherent in the technique, MRI usually detects the hydrogen of water in tissues of clinical patients. 4 Moreover, contrast agents, which usually are paramagnetic substances, for enhancing the MRI specificity as they locally reduce T 1 and T 2 relaxation times of the peripheral water molecules that are essential for clinical imaging. The contrast agents that predominantly reduce T 1 and T 2 cause increases and decreases in signal intensity, which brighten or darken images, respectively. The extent to which a contrast agent can change the T 1 or T 2 of solvent water is termed longitudinal (r 1 ) or transverse (r 2 ) relaxivity, which is defined as the slope of a linear fit for 1/T 1,2 versus concentration, respectively. Clinically, T 1 contrast agents exhibit higher spatial resolution than T 2 contrast agents. Moreover, signal draining sources in tissues often result in the false signal in the T 2 modalities but not T 1 modalities.
The contrast agent caused relaxation time decrease is related to their chemical properties. Therefore, activatable probes can be developed by using contrast agents that respond to specific changes in microenvironment, such as enzymatic activity, pH, temperature, and reducing environments. 4b,5 These probes should help to make a more specific diagnosis to diseases by monitoring various related physiological and pathological processes in realtime at the cellular and molecular level. 6 Therefore, the well-designed activatable probes that respond to the specific stimuli or change in concentration of the biomarker molecules are required. 7 Supramolecular chemistry has been defined as "chemistry beyond the molecule," and a multidisciplinary research field that mainly studies the noncovalent interactions in host-guest chemistry and self-assembly. 8 Supramolecular chemistry is involved in the design and implementation of functional chemical systems based on molecular components that are held together by noncovalent interactions. 9 In the past half century, supramolecular science as a multidisciplinary field has developed into a field that has expanded from basic science to applied science. 10 The dynamics and reversibility of noncovalent interactions have attracted great attention from researchers in the fields of chemistry, biology and materials science. 11 In addition to mastering and using this pre-organized structure, supramolecular chemistry also explores the design of self-organized systems, that is, a system that can spontaneously generate from its components into a well-defined and organized supramolecular structure through self-assembly. 12 A variety of complex functional supramolecular structures have been produced through the self-assembly process. 13 Supramolecular chemistry has been widely used in biomedical fields, as it utilizes the reversible and dynamic nature of noncovalent forces, 14 to construct a series of activatable events, such as activatable fluorescence imaging, 15 activatable photodynamic therapy, 16 activatable chemotherapy (supramolecular chemotherapy), 17 and activatable theranostics. 18 Similarly, supramolecular chemistry has also been used to perform activatable MRI, where an MRI contrast agent forms aggregates or host-guest complexes, accompanied by a change in the relaxation time due to slower tumbling. On encountering specific stimuli, the signal of the contrast agents is activated because of the altered state of the aggregates or the host-guest complex. In this review, we outline the advances in the understanding of the activatable MRI probes with supramolecular designs, the current challenges in the field, and the possible approaches to address these challenges. We discuss molecular recognition and self-assembly as two key topics in supramolecular chemistry, and describe a classification F I G U R E 1 The molecular structures of contrast agent 1 and the control contrast agent 1-ctrl, and the proposed chemical reaction following disulfide reduction and caspase-3/7-induced cleavage of DEVD peptide. GdNPs are Gd nanoparticles. Reprinted with permission. 27 Copyright 2014 by the Royal Society of Chemistry F I G U R E 2 Schematic diagram of PLA2 responsive liposomes with encapsulated Gd agents. Reprinted with permission. 32 Copyright 2014 by the Nature Publishing Group for MRI imaging based on self-assembly and molecular recognition.

MECHANISMS FOR MRI IMAGING
Currently, the most commonly used contrast agents are the gadolinium ion complexes. They have high relaxations because of their multiple unpaired electrons, big magnetic moments and long electron relaxation times. 19 According to the intrinsic properties and behavior of each contrast agent, different mechanisms must be considered when constructing 1 H-MRI activatable probes. The most widely used method for constructing T 1 contrast agents is to adjust the relaxation by preventing water from entering the coordination sphere of the paramagnetic metal, which decreases the value of r 1 . Therefore, this type of probes basically cannot be detected. Once contact with water molecules is restored, r 1 will significantly increase. In addition, the tumbling speed can be controlled by adjusting the r 1 value by connecting or detaching the contrast agent with the rigid support. In other cases, external triggering will cause the contrast agents to accumulate at the trigger point, thereby increasing local contrast. There are fewer activatable probes using T 2 contrast agent, and these probes are based on the release of encapsulated iron oxide nanoparticles or the accumulation of probes induced by precipitation.
In addition, chemical exchange saturation transfer (CEST) imaging is a recently developed imaging mechanism. 20 This method is based on probes having protons with chemical shifts that are very different from water protons, and these protons can be exchanged with water protons. This transfer is detected by the partial attenuation of the water signal. Using paramagnetic lanthanide complexes containing exchangeable protons can effectively enhance the CEST effect, because the downfield shifts of these exchanged protons relative to water protons are large.

MRI IMAGING BASED ON SELF-ASSEMBLY
Self-assembly is a "bottom-up" approach that constructs nanoscopic structures driven by noncovalent interactions between individual building blocks. 21 Based on multiple noncovalent interactions, molecular selfassembly has provided various thermodynamically stable nanostructures. 22 Owing to the weak and reversible noncovalent interactions between the building blocks, these assemblies are highly dynamic and responsive. 23 These properties have enabled self-assembled structures to be used in various applications, such as drug delivery, gene transfection, catalysis, and as sensors. 24 Therefore, materials constructing through self-assembly are good candidates for activatable probes. 25 Large payloads of contrast agents can be loaded into supramolecular structures for signal enhancement due to high local concentrations. For contrast agents based on Gd complex or iron oxide nanoparticles, 26 aggregation can cause increased relaxivity due to longer molecular tumbling time. 27 Moreover, these paramagnetic payloads may exhibit decreased relaxivity after entrapping into the inside of assembles. This is caused by the slower exchange of bulk water with the surrounding water of contrast agents 28 due to the low water permeability at the hydrophobic area. 29 For probes based on fluorine-19 based MRI ( 19 F MRI), the formation F I G U R E 3 (A) Schematic illustration of the GSH-controlled self-assembly that turns 19 F MRI signals "off", and the caspase 3/7-controlled disassembly that turns 19 F MRI signals "on". (B) The molecular structures of probe 2 and the 2-Scr probe.2-NPs are the nanoparticles formed by assembly of 2. Reprinted with permission. 30 Copyright 2015 by the American Chemical Society of assembles could induce the fast transverse relaxation of 19 F magnetic spin. 30 Responsive MRI probes based on self-assembly are mainly activated by enzyme, pH, redox, and small molecules, as described in the next section.

Enzyme-responsive MRI probes based on self-assembly
Enzymatic activity activated MRI probe is one of the main types of activatable probes, as enzyme dysregulation is a symptom of the disease. The presence of various active enzymes indicates the possibility of several kinds of cancer, and can reveal the progression of some pathologies, for example, atherosclerosis.
Caspase-3/7 is a cysteine protease that plays a crucial role in radiotherapy and chemotherapy-induced cell death of tumors. 31 Hence, its activity is a biomarker for apoptosis promoting antitumor therapies. In 2014, Rao et al.
developed a caspase-3/7 and glutathione (GSH) triggered macrocyclization of a Gd-based MRI probe 1 consisting of a Gd-DOTA complex, that was linked to a disulfide bond and a DEVD peptide substrate of caspase-3/7 ( Figure 1). 27 After macrocyclization, the probe assembled into nanoparticles and showed a change in r 1 from 10.2 to 19.0 mM -1 s -1 at 1 T in solution because of the increased tumbling time. In the HeLa cell line, probe 1 showed a much higher response to caspase-3/7 activity than 1-ctrl, which cannot macrocyclize and self-assemble.
Phospholipase A 2 (PLA 2 ) can catalytically hydrolyze the sn-2 acyl bond of glycerophospholipids and is a biomarker for many diseases, including acute sepsis, atherosclerosis, pancreatitis, and some cancers. In 2014, Cheng and Tsourkas reported the construction of liposomes loaded with gadoteridol, a clinically approved Gdbased MR contrast agent, for PLA 2 responsive MRI imaging ( Figure 2). 32 Gadoteridol in the liposomes exhibited a low T 1 -weighted signal due to of the decrease in Recently, 19 F MRI has gained increased attention owing to various properties that complement 1 H MRI. 33 First, the F I G U R E 6 (A) Schematic illustration of pH-responsive 19 F-MRI probes of ionizable copolymers. (B) The molecular structure of typical copolymers 5, which contain different pH-responsive and 19 F reporter part, with their respective pK a values and 19 F chemical shifts shown in parentheses. Reprinted with permission. 38 Copyright 2013 by Wiley-VCH Verlag GmbH & Co. KGaA F I G U R E 7 Molecular structures of 6-Gd and 6-Gal, and the schematic illustration of disassembly of their coassemblies triggered by GSH. Reprinted with permission. 40 Copyright 2020 by American Association for the Advancement of Science natural abundance of 19 F isotope is 100 %, and its sensitivity and signal-to-noise ratio in NMR is comparable to that of 1 H. Second, compared to 1 H MRI, the use of 19 F MRI exhibits negligible endogenous signals in the body. Therefore, 19 F MRI images show no detectable background signal because the signal can only come from exogenous probes. This allows for the quantification of the probe concentration in body through imaging. Thirdly, 19 F MRI can also be used to design activatable imaging systems, whose signal can be activated by external stimuli.
In 2015, Liang et al. reported the construction of the 19 F MRI probe 2 for imaging caspase 3/7 activity in vivo. 30 In the presence of GSH, the probe can undergo reduction and assemble into nanoparticles, which have no detectable MRI signal. Subsequently, when encounter caspase 3/7, the nanoparticles disassembled and their MRI intensity F I G U R E 8 Schematic for stimuli-responsive liposomes that respond to small molecular targets for multimodal detection. Reprinted with permission. 43 Copyright 2016 by the American Chemical Society increased significantly (Figure 3). By employing this strategy, the 19 F MRI probe 2 has been used for imaging caspase 3/7 activity at the cellular level and in vivo by using a 14.1 T magnetic field. In contrast, the control probe 2-Scr forms aggregates under the stimulus of GSH but shows undetectable MRI signal in zebrafish, as it cannot be cleaved by caspase 3/7. By altering the peptide part, they also developed GSH induced assembly and legumaininduced disassembly to detect the enzyme activity using the similar strategy. 34 These probes enabled the study of the targeted enzymes and the detection of the presence of GSH.
In 2016, Liang et al. 35 designed a fluorine responsive hydrogel that could detect the activity of alkaline phosphatase (ALP) and tyrosine kinase using MRI imaging ( Figure 4). ALP causes the hydrolysis of 3b to 3a, which forms a hydrogel with undetectable 19 F MRI signal. Moreover, tyrosine kinase results in disassembly of the hydrogel of 3a, accompanied by the increased 19 F MRI signal.

pH-Responsive MRI probes based on self-assembly
The pH values in tumor cells are lower than in healthy cells. 36 Therefore, the probes to image acidic (pH values 5.5 to 6.8) area can be used as indicator for the existence of cancer.
In 2016, Lee et al. developed the pH-responsive aggregates of the polymer 4, which is encapsulated with iron oxide nanoparticles for use in MRI imaging ( Figure 5). 26 Polymer 4 contains the hydrophilic head mPEG, and a polypeptide part with side chains having amine-terminated dopamine and pH-sensitive 2-(dibutylamino)ethylamine group. The aggregates of polymer 4 can disassemble at acidic environment, causing the release of iron oxide nanoparticles and an enhancement in the contrast of T 2 . Using this modification, the acidic tissues resulting due to cerebral ischemia can be visualized in a rat model.
In 2013, McMahon et al. developed a CEST based biosensor for detecting cell viability in subcutaneously transplanted hydrogel-encapsulated cells. The low extracellular pH values were used for probing cell death. The CEST contrast of the l-arginine probe significantly decreases at a more acidic pH area than normal tissue. They filled larginine containing liposomes and hepatocytes into alginate microcapsules. The system can monitor cell viability real-time by analyzing the change in the CEST contrast caused by local acidic pH. 37 In 2013, Gao et al. 38 reported the construction of several multichromatic pH-activatable 19 F probes, which formed micelles when their pH values were higher than the pK a values ( Figure 6). When the micelles disassembled under lower pH conditions, the fluorine signal during the MRI was turned on. According to their pK a , the probes can detect specific transitions at pH 6.5, 5.5, and 4.5.

Redox-responsive MRI probes based on self-assembly
In order to detect changes in the redox state, manganese probes are usually used because the trivalent or tetravalent manganese complexes or salts are stable, can be widely used and can be easily reduced to the Mn(II) state. It is F I G U R E 9 Molecular structure of 7 and the cartoon image presenting 7 complexing with Ca 2+ . Reprinted with permission. 47 Copyright 2016 by the American Chemical Society better used as a T 1 contrast agent than the salt used as Mn(III) or Mn(IV).
In 2016, Kim et al. reported a T 1 /T 2 redox responsive contrast agent using iron oxide nanoparticles coated with a Mn 3 O 4 shell. 39 In the absence of GSH, the relaxivity of the contrast agent is very low. However, in the presence of GSH, the shell decomposes into Mn 2+ and exposes iron oxide nanoparticles, thereby increasing r 1 and r 2 . The feasibility of this imaging method to create a reducing environment in tumors was evaluated both in vitro and in vivo.
In 2020, Ye et al. developed a liver-targeted and GSH-responsive trimodal probe for rapid evaluation of lipopolysaccharide-induced acute liver inflammation. 40 The probe formed through the coassembly of 6-Gd and 6-Gal, which can image GSH depletion through 1 H-MRI, 19 F-MRI and fluorescence imaging (Figure 7). The coassembled probe exhibited high r 1 relaxivity, low fluorescence and 19 F-MRI signal. Upon interaction with GSH, 6-Gd and 6-Gal were cleaved and the aggregates rapidly disassembled into small molecules, accompanied with a substantial decrease in r 1 relaxivity and enhancements in fluorescence and 19 F-MRI signal. The authors noninvasively visualized the lipopolysaccharideinduced acute hepatitis and its remediation in mice after treatment with dexamethasone, an anti-inflammatory drug.
Other redox-responsive MRI probes include Gd (III) loaded in polymer nanoparticles with redox labile bonds. After the unstable bond is reduced, Gd (III) is exposed to water with an increase in r 1 . 41 Rao et al. used a macrocyclization reaction to construct Gd-labeled nanoparticles in situ. After the nanoparticles responded to GSH, the r 1 relaxation value of the nanoparticles could increase by about 60%. 42

Small molecule-responsive MRI probes based on self-assembly
In 2016, Lu et al. 43 reported a method for the multimodal fluorescent and MRI based detection of cocaine (Figure 8). In this approach, magnetic beads are first modified by DNA aptamers conjugated to PLA 2 . Next, liposomes are The liposomes can also be used as uranin fluorophores to achieve multimodal detection, and the method can potentially be extended to other analytes using other DNA aptamers.

MRI IMAGING BASED ON MOLECULAR RECOGNITION
Molecular recognition is a core concept in supramolecular chemistry, 44 and it is used to describe the specific association of molecules through various noncovalent forces including hydrogen bonding, hydrophobic effect and van der Waals forces. 45 In nature, molecular recognition by proteins is fundamental to almost every biological process. During the past half a century, multiple advances have been made in understanding molecular recognition in parallel with the invention of numerous synthetic receptors. 46 After complexation with supramolecular hosts, the signal from MRI probes change due to the different environments in bulk water. In the next section, we describe molecular recognition based responsive MRI probes that are mainly activated by ions and small molecules.

Ion-responsive MRI probes based on molecular recognition
In 2016, Angelovski et al. reported the development of the dendrimeric MRI probe 7 that has an affinity for binding to Ca 2+ (Figure 9). 47 After adding Ca 2+ into the solution, the r 1 and r 2 of probe 7 increased by ∼70% and ∼360%, respectively. The change in the T 2 /T 1 can be recorded quickly in the absence and presence of Ca 2+ .
In 2009, Chang et al. developed a new class of copper responsive MRI probes 8-12. 48 These indicators are comprised of a Gd 3+ complex part for producing signal and a thioether-rich receptors part for complexing copper. When there are no copper ions, the binding of the internal spherical water to the Gd 3+ chelate is restricted, which causes a low r 1 . The addition of Cu + to the solution of contrast agents 8-11 and the addition of Cu + or Cu 2+ to the contrast agent 12 can result in a significant increase in relaxivity. Compared with other competing metal ions at cell concentrations, these probes are highly selective for copper ions. The T 1 -weighted MRI images of these probes can visualize changes in copper levels under clinically used field strengths ( Figure 10). In 2012, Que et al. further reported that in the presence of other cations, modification of Gd-DO3A with thioether chelation can selectively bind to Cu + , resulting in a significant increase in r 1 . 49 The probe has successfully tested in a copper-exposed cell line, which is a model of Menkes disease.
In 2012, Logothetis et al. synthesized a kind of Ca 2+sensitive probes for studying the functional role of Ca 2+ in the brain in a non-invasive manner by using MRI (Figure 11). 50 In buffer solutions or physiological fluids, the probe can selectively bind to Ca 2+ other ions. Adding Ca 2+ to the solution of the probes results in remarkable increase in their T 1 relaxivities, which is caused by the enhanced hydration state and the slower rotational movement. The probes have been used for imaging Ca 2+ in the extracellular fluid of the brain.
In 2014, they developed a Gd 3+ -based MRI probe sensitive to Ca 2+ and tried to use it as a functional marker for use in the body. 51 First, they used a three-dimensional cell culture model to quantify the relaxation response to the probe. Next, they studied the changes in the function of primary glial cells after using the probe. The monitoring of intracellular Ca 2+ levels showed that although the Ca 2+ level was reduced, the transport of Ca 2+ through the plasma membrane was not affected.
In 2016, Meade et al. reported a Ca 2+ responsive MRI probe 13 to image the phenomenon of Ca 2+ flux in the central nervous system. 52 The probe 13 has four ester groups, which can increase cell uptake. Moreover, the ester groups can be convert to carboxyl groups by intracellular esterase. Therefore, the MRI signal of 13 respond to intracellular but not extracellular Ca 2+ (Figure 12). The ability of this probe has potential to real-time visualize Ca 2+ flux in vivo.

Small molecule responsive MRI probes based on molecular recognition
In 2014, Jasanoff et al. demonstrated the use of an MRI probe responding to dopamine for imaging brain activity. 53 They used a protein, BM3h-9D7, which can specifically complex with dopamine, as the MRI probe of dopamine. The probe can be used to monitoring the process of changes of dopamine caused by electrical and chemical stimulations. This work demonstrates that the quantitative molecular MRI has potential to reveal various phenomena in neuroscience in a noninvasive manner. However, the paramagnetic probes for T 1 -weighted MRI are effective only at micromolar concentrations and will perturb neurochemistry. In 2019, they reported T 1 -weighted MRI probes of superparamagnetic iron oxide nanoparticles conjugated to designed dopamine analogs and BM3h-9D7. 54 The sensors exhibit significant relaxivity changes In 2020, they further developed a new strategy for small molecules responsive MRI imaging. 55 They used a potent vasodilator, pituitary adenylate cyclase activating polypeptide (PACAP), which can cause vasodilation and accompanied enhanced MRI signal (Figures 13a and 13b). After attaching to designed analogs of target molecules of interest, PACAP can complex with a protein domain that selectively binds the targets and the designed analogs of the target (Figure 13c). In the presence of the target, the modified PACAP is released and can be detected by MRI.
In 2019, O'Reilly et al. reported the construction of a 19 F MRI probe 14 with a trifluoromethyl part providing signal and an adamantine part for complex with (2hydroxypropyl)-β-cyclodextrin (HP-β-CD). 56 After forming complexes with HP-β-CD, the environment of probe 14 was varied and therefore the fluorine mobility changed, which can be measured through MRI imaging ( Figure 14). Additionally, this work demonstrated the possibility to modulate the 19 F MRI signal through host-guest complexation, and has potential to expand to monitor the pharmacokinetics in vivo.

SUMMARY AND PERSPECTIVES
In summary, the development of activatable MRI probes that respond to specific stimuli for aiding the diagnosis of diseases is garnering increasing interest. Activatable MRI probes can be designed using a supramolecular strategy. Herein, we summarized the achievement of activatable MRI probes that are designed for molecular recognition and self-assembly. Through the self-assembly approach, researchers have developed MRI probes that can be activated by stimuli including enzymatic activity, pH values, and redox potential. Through the molecular recognition approach, MRI probes that respond to small molecules, such as metal ions and dopamine, have also been reported. These examples illustrate various advantages of supramolecular strategy for activatable MRI imaging, including the ease of construction, sensitive response to external stimuli, and the easy adapt to other MRI probes. However, there is yet room for further development of several aspects of activatable MRI imaging. First, new biosignaling-based activation mechanisms need to be developed. As a result, more biomarkers are expected to be used to activate MRI signals. This will allow for imaging the concentrations of different kinds of targets without the need for further dedicated synthesis. To develop an MRI probe with enough selectivity to specific stimuli, we need to consider both supramolecular activation strategies and receptor design. Second, most of the reported MRI probes based on supramolecular design are dedicated to imaging the area of cancer. The MRI probes activated by the environment of other important diseases such as cardiovascular diseases need to be further explored. It is hoped that the supramolecular strategy would reasonably prompt the development of activatable MRI imaging, based on its demand.

C O N F L I C T O F I N T E R E S T
The authors declare no conflict of interest.

A C K N O W L E D G M E N T
This work was supported by NSFC (51873090 and 31961143004) and the Fundamental Research Funds for the Central Universities, which are gratefully acknowledged.