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Chem Rev. Author manuscript; available in PMC 2020 January 23.
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Published in final edited form as:

Chem Rev. 2019 January 23; 119(2): 957–1057. doi:10.1021/acs.chemrev.8b00363.

Chemistry of MRI Contrast Agents: Current Challenges and New

Frontiers
Jessica Wahsner#, Eric M. Gale#, Aurora Rodríguez-Rodríguez, and Peter Caravan*
Athinoula A. Martinos Center for Biomedical Imaging and the Institute for Innovation in Imaging,
Department of Radiology, Massachusetts General Hospital and Harvard Medical School,
Charlestown, MA 02129, USA
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# These authors contributed equally to this work.

Abstract
Tens of millions of contrast enhanced magnetic resonance imaging (MRI) exams are performed
annually around the world. The contrast agents, which improve diagnostic accuracy, are almost
exclusively small, hydrophilic gadolinium(III) based chelates. In recent years concerns have arisen
surrounding the long term safety of these compounds, and this has spurred research into
alternatives. There has also been a push to develop new molecularly targeted contrast agents or
agents that can sense pathological changes in the local environment. This comprehensive review
describes the state of the art of clinically approved contrast agents, their mechanism of action, and
factors influencing their safety. From there we describe different mechanisms of generating MR
image contrast such as relaxation, chemical exchange saturation transfer, and direct detection and
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the types of molecules that are effective for these purposes. Next we describe efforts to make safer
contrast agents either by increasing relaxivity, increasing resistance to metal ion release, or by
moving to gadolinium(III)-free alternatives. Finally we survey approaches to make contrast agents
more specific for pathology either by direct biochemical targeting or by the design of responsive
or activatable contrast agents.

Graphical Abstract
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*
Corresponding author: [email protected].
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1. Introduction
Gadolinium(III) based contrast agents (GBCAs), as a class, are one of the most successful
examples of inorganic drugs, along with the platinum anticancer compounds and the
technetium-99m radiopharmaceuticals used for cardiac and bone imaging. This success
comes from the diagnostic and prognostic information that these agents provide, as well as
their very favorable safety profile when compared to other imaging agents or drugs. GBCAs
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are used in about 40% of all magnetic resonance imaging (MRI) exams and in about 60% of
neuro MRI exams. This represents about 40 million administrations of GBCAs worldwide.1

The growth of MRI and contrast-enhanced MRI has been remarkable. When one of us
reviewed the field in 1999 we noted that since the approval of Gd-DTPA in 1988, about 30
metric tons of gadolinium metal ion had been cumulatively administered to patients
worldwide over that 11 year period.2 Now almost 50 tons of gadolinium are administered
annually. The market for GBCAs is over one billion dollars per year. What has driven this
growth?

GBCAs have been so successful because they provide essential diagnostic information that
often cannot be obtained with other noninvasive techniques. A prime example is using
GBCAs to detect blood brain barrier (BBB) disruption. GBCAs do not cross the BBB, so
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contrast enhancement of the brain can arise from pathologies such as multiple sclerosis,
primary or metastatic cancer, or stroke. The absence of contrast enhancement can also
enable a differential diagnosis when other symptoms are considered. GBCAs also detect
increased vascular permeability associated with lesions outside the brain as well, for
example in breast cancer detection and staging. GBCAs are also widely used to image the
blood vessels to detect blockages or aneurysms, or to guide surgical interventions. One can

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also exploit the kinetics of contrast enhancement after injection, for example to measure
regional perfusion of the heart.3
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Another factor in the success of GBCAs is that their effect is immediate. Unlike in nuclear
medicine where the patient receives the radiopharmaceutical and then waits a period before
imaging, in MRI the contrast agent is administered while the patient is in the scanner and
diagnostic images appear within minutes. This practical aspect allows more patient
throughput and facilitates workflow in radiology practice. Unlike radioactive tracers used in
nuclear medicine, GBCAs are shelf-stable and do not need to be synthesized on demand.
Moreover there is no ionizing radiation associated with GBCAs or MRI in general. A further
factor is that GBCAs shorten the T1 of protons and this allows for faster imaging (higher
throughput) and for imaging that is less sensitive to artifacts from motion (better quality
images).
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Gadolinium is not the only element that can be used to generate MR image contrast. Iron
oxide nanoparticles and a manganese(II) complex have been approved for imaging the liver,
although these were not commercially successful. In our 1999 review we focused solely on
gadolinium(III) complexes as contrast agents. At that time it was clear that the Gd(III)
complexes would dominate the field. In this current review we focus more broadly. In the
intervening time a number of changes have occurred to cause us to cast a wider net.

For 18 years following GBCA approval for human use, these compounds were considered to
be among the safest pharmaceuticals. Indeed the rate of immediate adverse events is very
low (<1 per thousand injections) and usually mild with severe adverse events occurring at
about once per 40,000 injections.4 However in 2006 GBCAs were associated with a
devastating and potentially fatal condition called nephrogenic systemic fibrosis (NSF).5–6
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NSF occurs in patients with poor kidney function and its onset can occur months after the
last GBCA administration. More recently it has been recognized that some fraction of the
injected gadolinium can remain in the body for long periods, although the chemical form of
Gd(III) and its whole body distribution is still not well understood. Repeat usage of GBCAs
can result in a buildup of residual Gd(III) to the point where it can be detectable by MRI or
other methods.7 Together, these findings have led to renewed interest in finding alternatives
to using gadolinium(III) for MR contrast.

New techniques for contrast agent detection have also emerged or been improved. In a
seminal 2000 paper Ward and Balaban proposed generating image contrast by saturating
exchangeable hydrogen resonances (e.g. N-H protons) which then chemically transfer this
saturation to bulk water resulting in signal loss.8 There have been a number of clever
approaches to exploit this mechanism involving small molecules, metal chelates, and
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nanoparticles. Direct detection of nuclei such as C-13 or F-19 had been limited because of
very low sensitivity when compared with imaging water. However gains in hyperpolarization
technology have enabled imaging of hyperpolarized C-13 labeled molecules in humans.9
Pyruvate can be hyperpolarized, injected into the body and then quantified, along with its
metabolic transformation into alanine and lactate. Imaging pyruvate metabolism may be
useful in staging cancers. Fluorine-19 in the form of perfluorocarbon emulsions can be used

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to label cells and 19F MRI can be used to image the location and concentration of these cell
therapies once they are injected into the body.10
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The ability of GBCAs to delineate pathology is based mainly on non-specific distribution.

Most GBCAs have an extracellular distribution and are eliminated via the kidneys.
Pathology is detected because of altered anatomy (e.g. a narrowed blood vessel) or
physiology (e.g. leaky blood brain barrier). GBCAs that distribute to the liver can detect
liver lesions that don’t take up the contrast agent. However there is a great potential to move
beyond passive distribution and create contrast agents that are biochemically targeted and
potentially more specific to disease.

Contrast agents are detected indirectly by their effect on bulk water molecules. The
magnitude of this effect is strongly influenced by the chemical properties of the contrast
agent and can be potentially be modulated to produce a stronger signal in certain
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environments. Activatable contrast agents, whose signal is altered by pH, enzymatic activity,
temperature change, or ion flux, represent an exciting and growing area of research.

The goals of this review are to provide an overview of the state of art of approved MRI
contrast agents, their applications, and the risks associated with them; to provide an
understanding of the molecular mechanisms that give rise to MR contrast from relaxation
and exchange; to review the coordination chemistry of these agents and the factors
responsible for designing an agent safe for human use. We then move from this foundation
to review new gadolinium(III)- and non-gadolinium(III) based contrast agents, molecularly
targeted agents, and activatable agents. The review should provide the reader with an
understanding of the state of the art of MRI contrast agents, the current challenges in the
field, and the strategies employed to overcome these challenges.
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We start with a description of contrast agents that have been approved for human use. Since
these are so well studied, they also the form the basis of descriptions of mechanism of action
as well as stability and safety. The field of MRI contrast agents has grown tremendously in
the last decades. It has not been possible to cover all of the outstanding chemistry reported.
For reasons of scope, we focus primarily on discrete molecular entities (small molecules,
metal chelates). While there is some discussion of nanoparticle based agents, this is limited
to formulations that have been studied in humans and some exemplary representatives of
targeting and/or high relaxivity. We also focus on contrast agents, molecules that generate
image contrast in MRI, and provide only a brief overview of agents that are directly detected
like hyperpolarized nuclei or perfluorocarbon emulsions.

2. Standard of care and new frontiers

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2.1 Applications
MR contrast agents are an absolutely integral component of modern radiology. The first MR
contrast agent was made available in 1988 for imaging blood-brain barrier abnormalities and
since then contrast enhanced MRI has played an increasingly important role is diagnostic
medicine.11 Today, there have been a dozen FDA approved MR contrast agents and 8 of
these agents are still commercially available in the United States. Contrast enhanced MRI is

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now routinely used for imaging lesions in the central nervous system, breast, and abdomen,
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for angiographic imaging, cardiac imaging, and articular imaging, amongst numerous
additional applications. Tens of millions of contrast enhanced examinations are ordered
annually worldwide.

All 8 of the MR contrast agents commercially available in the United Stated are small
molecule Gd(III) complexes. Seven of the commercially available agents received their
primary indication for imaging lesions of the central nervous system (CNS), but one agent
(Gd-EOB-DTPA) received FDA approval for liver imaging applications. There are four MR
contrast agents that have FDA approval but are no longer currently marketed. The Gd(III)-
complex MS-325 was approved for angiographic (blood vessel) imaging. A Mn(II) complex
(Mn-DPDP) and a superparamagnetic iron-oxide nanoparticle (SPION) formulation
(ferumoxide) were approved for liver imaging, and an oral SPION formulation was approved
for gastrointestinal imaging (ferumoxsil).
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The seven commercially available Gd(III)-based MRI contrast agents with indications for
CNS lesions are administered as an intravenous bolus. These contrast agents are classified as
extracellular fluid (ECF) contrast agents. They rapidly extravasate from the bloodstream into
the tissue interstitial, extracellular spaces and are rapidly eliminated from the systemic
circulation via filtration through the kidneys. Two of these agents are partially excreted
through the liver and into the bile and feces.

The contrast enhancement observed after injection reflects the distribution of the
exogenously administered agent, and extracellular agents can be ideal for identifying
pathologic tissue. For example, tumors are typically characterized by hyper-vascularity,
compromised endothelium, and/or an underdeveloped lymphatic drainage system and thus
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temporarily retain greater concentrations of the contrast agent than healthy tissue and for
longer periods of time. The contrast agent thus provides conspicuous contrast enhancement
of the tumor in the minutes after injection. Important information about the tumor
vascularity and perfusion can be learned by acquiring scans during the contrast agents first
pass through the arteries or by dynamically monitoring accumulation and clearance of the
contrast agent from the tumor. In the seconds immediately following bolus injection,
extracellular MRI contrast agents can also provide high-resolution imaging of the blood
vessels to detect vascular pathologies such as stenoses, occlusions, and aneurisms. Contrast
agents that are partially eliminated via the hepatobiliary system provide strong contrast
enhancement of liver parenchyma as the agent transits the liver in the minutes after the blood
signal has largely returned to baseline. This contrast is useful for differential diagnosis of
liver malignancies which are devoid of normal hepatocytes.
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2.2 New Directions

Contrast agents with biochemical specificity—The clinically available ECF agents
are specifically designed to passively move through patients with little or no interactions
with proteins or cells.12 The signal observed is strictly reflective of the agent’s distribution.
ECF agents provide superb contrast to differentiate anatomical structures or to characterize
pathophysiology, but do so in a nonspecific manner. However, new contrast agents with the

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capability to detect or quantify pathologic biomarkers could revolutionize the specificity

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with which MRI can be used to detect, characterize, and quantify pathologic tissue.

There are two general approaches to increased biochemical specificity. The first is to couple
the contrast agent to a targeting vector to localize the agent to a specific protein or cell type.
However the concentration of a monomeric contrast agent needed for detection is at least in
the micromolar range. Thus abundant targets need to be selected, large assemblies of the
imaging reporter must be used, or some other mechanism of target accumulation must be
exploited. The second approach is to modulate the contrast generating signal in response to
some stimulus. For example the hydration state of the contrast agent may change with pH
resulting in increased or decreased signal. Or the contrast agent may be chemically
transformed by the action of a specific enzyme, resulting in a change in signal. Sections 5
and 6 will highlight innovate approaches towards biochemically specific targeted and
activatable MRI contrast agents, respectively.
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3. Background and Theory

3.1 Classification of contrast agents: Biodistribution and applications
MR contrast agents can be administered either (1) intravenously; (2) orally or (3) by
inhalation. Intravenous MR contrast agents may be categorized in 3 types: (a) extracellular
fluid (ECF); (b) blood pool; and (c) target/organ-specific agents (Figure 1).

3.1.1 Intravenous MRI contrast agents—a) Extracellular fluid (ECF) agents are
distributed between the intravascular and cellular space. ECF agents are low molecular
weight chelates and after intravenous injection, they travel to the heart and then out to the
systemic arteries, leaking into the extravascular, extracellular space. They are rapidly
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eliminated from the body through the renal pathway (see Figure 2). ECF agents are the most
commonly used contrast agents and represent >98% of all contrast agents sold in the United
States.13 They are generally employed to image arterial abnormalities and to detect altered
tissue endothelium, e.g. disrupted blood brain barrier. ECF agents approved for clinical use
all consist of a nine-coordinate Gd(III) ion chelated by an octadentate polyaminocarboxylate
ligand with a water co-ligand (Figure 3). The ECF agents approved for human use are listed
in Table 1. Like all drugs, contrast agents have a commercial name like “Dotarem®” and a
generic name, in this case gadoterate meglumine. There is also a chemical abbreviation, and
in this case Gd-DOTA or [Gd(DOTA)(H2O)]−. In the literature the reference to the
commercial name can be confusing. In the past, the ECF agents were dominantly sold by a
single supplier such that gadoterate meglumine = Dotarem. But now there are increasing
numbers of generic products. For example GE Healthcare now sells its own version of
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gadoterate meglumine which is marketed as “Clariscan®” which has the same Gd-DOTA
chelate that is used in Dotarem®. Clariscan was also the name given to an iron oxide
nanoparticle formulation NC100150 that underwent clinical trials in the 1990s, but was
never approved for commercial use. In general it is best to avoid the use of commercial
names to avoid confusion over the compound involved and to avoid product promotion for a
particular commercial entity.

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b) Blood pool agents are compounds that are, after injection, restricted to the intravascular
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space and which provide high contrast for imaging the arteries and veins. In the 1990s there
were many blood pool agents under development. There were three main strategies
employed, all of which resulted in compounds that underwent human clinical trials: (1) low
molecular weight compounds that bind noncovalently to human serum albumin upon
intravenous injection which restricts distribution to the intravascular space. An example is
MS-325 (gadofosveset trisodium which had commercial names of Angiomark, Vasovist, and
Ablavar) (Figure 3). MS-325 was 80-90% bound to HSA but the unbound fraction could be
eliminated through the kidneys.16–21 MS-325 was approved for commercial use but is no
longer commercially available. The compound B22956 (Figure 4) was another albumin-
binding Gd-DTPA based agent that progressed to Phase 2 clinical trials.22–26 (2) Large
discrete Gd(III)-based compounds that were too large to rapidly extravasate across the
endothelial barrier, but still small enough to be filtered by the kidney. The dendrimer
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Gadomer (aka Gadomer-17) (Figure 4) was a 17 kDa dendrimer with 24 macrocyclic

Gd(III)-chelates that advanced to Phase 2 clinical trials.27–30 Gadomer combined increased
size with a high Gd(III) payload for high relaxivity. The compound P792 (Figure 4) was a
single Gd-DOTA derivative the chelate at the barycenter and 4 large hydrophilic branches to
increase the hydrodynamic radius.31–34 This compound was also used in Phase 2 clinical
trials. (3) Small iron oxide nanoparticles that had a high T1 relaxivity. These compounds are
truly intravascular and have long blood circulation times. The nanoparticles are not cleared
from the body, rather they eventually accumulate in the liver and spleen and the iron is
incorporated into the body’s pool of iron. Iron oxide particles that underwent Phase 2 and/or
Phase 3 angiography clinical trials were NC100150,35–36 VSOP-C184,37–38 and
AMI-227.39–43 None of these was ultimately approved for human use. The SPION
ferumoxytol was approved for use as an intravenous iron replacement therapy in anemic
patients. Ferumoxytol has excellent T1 relaxivity and has been used off-label at some
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institutions as a blood pool agent in humans. Off-label means that the compound is not being
used for its approved packaging label (in this case to treat anemia).

The lack of approved and commercially available blood pool agents is driven by market
demand. In the 1990s when most of these agents were being developed, the MR scanner
performance was insufficient to support dynamic contrast enhanced MR angiography
(MRA). Dynamic MRA involves very rapid injection of a contrast agent and rapid imaging
to get an image of the contrast agent as it traversed the systemic arteries after injection but
before it arrived in the venous circulation. However power injectors (for reproducible rapid
injection) and higher performing scanners with faster gradients (for faster imaging) enabled
contrast enhanced MRA to be routinely performed with ECF agents, and there is now much
lower demand for dedicated blood pool agents.
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c) Organ-specific agents are capable of targeting specific organs or tissues, for example the
liver, spleen, or lymph nodes. ECF agents are eliminated via the kidneys. Altering the
elimination pathway to include the liver can enable liver-specific imaging. Table 3 lists
examples of contrast agents that are approved for commercial use or were studied in human
clinical trials. For instance, the approved MRI contrast agent gadoxetic acid is rapidly taken
up by hepatocytes and can therefore be used to image the liver and biliary system.44

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Gadobenate can also be used for liver imaging but its hepatocyte uptake is much lower than
that of gadoxetic acid.45 A Mn(II)-based example is the agent mangafodipir (Mn-DPDP)46,
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that dissociates after injection to the free Mn(II) ion. The Mn(II) is then rapidly taken up by
hepatocytes, cardiomyocytes, and pancreatic cells, and can be employed to image the liver,
myocardium, or pancreas. Organ-specific agents that are based on iron oxide particles
exhibit a different distribution. SPIONs are imported into the cells of the reticuloendothelial
system through phagocytosis, which enables selective access to the liver, spleen, lymph
nodes, tumor associated macrophages, and bone marrow.47 Depending on the SPION size
and coating, different tissues can be targeted. For instance ferumoxide48 and ferucarbotran49
were both rapidly taken up by the Kuppfer cells in the liver and enabled liver-specific
imaging. Ferumoxtran-10 has a longer circulating time and can be used for blood pool
imaging, but with time also significantly accumulates in lymph nodes which enables cancer
staging applications.50 None of the approved SPIONs, nor mangafodipir are commercially
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available anymore, and to the best of our knowledge clinical development has been
discontinued.

3.1.2 Oral contrast agents—Oral contrast agents are orally administered and are
suitable for imaging the gastrointestinal tract (GI) imaging. Gd(III)- and Mn(II)-based
agents, SPIOs, barium sulfate suspensions as well as fruit juice rich in manganese (e.g.
blueberry pineapple juice) have been investigated as oral contrast agents.51–56 The following
Table 4 summarizes the OCAs that are currently available for clinical application. These are
not widely used.

3.1.3 Ventilation agents—Ventilation agents are contrast agents that are inhaled to
improve the diagnostic value of MRI for the lungs. Gadolinium(III)-based aerosols and
oxygen gas are paramagnetic and after inhalation they can be used to estimate ventilation in
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the lungs by its effect on lung water T1. Direct detection is also used with hyperpolarized
gases (3He, 129Xe) and with inert perfluorinated gases such as SF6.57

3.2 Classification of contrast agents: Biophysical mechanism of action

Contrast agents can also be classified in terms of their contrast generating mechanism of
action. We have grouped contrast agents into four major classes (1) paramagnetic; (2)
superparamagnetic; (3) chemical exchange saturation transfer (CEST); and (4) direct
detection.

3.2.1 Paramagnetic contrast agents—Paramagnetic contrast agents are by far the

most prominent MRI contrast agents. They are also called positive contrast agents because
they increase MR signal in regions where they distribute.
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Paramagnetic contrast agents comprise a metal ion that has unpaired electrons. Gd(III) with
its half-filled f7 shell and high spin Mn(II)- and Fe(III)-complexes with their half-filled d5
shells all exhibit a large magnetic susceptibility compared to other metal ions. The
symmetric S electronic ground state also endows these compounds with a relatively slow
electronic relaxation rate which is necessary to promote strong nuclear relaxation. Of all the
potential metal complexes that could be imagined, discrete Gd(III) chelates have been the

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most successful paramagnetic contrast agents so far and clearly dominate the contrast agents
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used in the clinic. We will start with a description of approved contrast agents as this will
inform discussions of newer agents.

Clinically used Gd(III)-based contrast agents (GBCAs) all utilize an octadentate

polyaminopolycarboxylato-based ligand and have a ninth coordination site available for
water ligation. The coordinated water ligand can be rapidly exchanged with bulk water
molecules. GBCAs that have been approved for clinical use are illustrated in Figure 3. As
outlined above, some of these are no longer available for commercial.

To date, only two manganese-based contrast agents have ever been approved: Mn-DPDP
(Figure 3) and an orally administered contrast agent that consists of liposomal encapsulated
manganese chloride (LumenHance).58
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Contrast agents containing Gd(III) shorten the observed longitudinal (T1) and the transverse
(T2) relaxation time of water protons in their vicinity. The rate constants corresponding to
the T1 or T2 relaxation times are defined as 1/T1 and 1/T2, respectively, which are generally
referred to as relaxation rates. In this review, we will use the general term relaxation rate
when referring to the rate constant of spin relaxation. On T1-weighted scans, tissue with
short T1 appears bright and thus T1-shortening contrast agents generally make signal bright.
On the contrary, T2-weighted scans show tissue with long T2 as being bright, so T2-
shortening agents reduce signal. The extent to which a contrast agent can change the T1 or
T2 of solvent water is termed relaxivity r1 or r2, respectively; this is sometimes referred to as
longitudinal (r1) and transverse (r2) relaxivity. Relaxivity is defined by equation 1, where
(1/Ti0) is the inherent relaxation rate of the tissue, (1/Ti) is the relaxation rate in the presence
of contrast agent (ri) and [CA] is the concentration of the contrast agent.
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1 1
= + ri[CA]; i = 1, 2 (1)
Ti T0
i

Low molecular weight Gd(III) complexes have similar r1 and r2 values in water that have
very little variation with field at magnetic fields generally used for MRI (Figure 5).

The relaxation times of water hydrogen nuclei in pure water are very long. But in different
tissues the relaxation times can be much shorter due to the interaction of water with
macromolecules and also because of the presence of endogenous paramagnetic species such
as ferritin.60 In the body, water T2 is generally 5 – 20 times shorter than T1. As a result, the
effect of a Gd(III) contrast agent will be much more pronounced on T1. This is illustrated in
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Figure 6 where the endogenous relaxation rates of grey matter at 1.5 T are plotted (solid).
Addition of 1 mM Gd-DOTA (r1 = 3.6 mM−1s−1, r2 = 4.3 mM−1s−1) will increase the 1/T1
and 1/T2 by 3.6 and 4.3 s−1, respectively. Because of the low baseline T1 relaxation rate, the
effect of the contrast agent on 1/T1 is much larger (+ 428 %) than on 1/T2 (+ 41 %). Because
of this effect, Gd(III) based agents are often referred to as T1-contrast agents because on a
percentage basis they have a much larger effect on T1.

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Figure 7 is an exemplary T1 weighted image of the brain of a patient with glioblastoma and
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shows the effect of the T1 contrast agent gadoteridol at 3 T. Twenty minutes after injection
of gadoteridol, the tumor becomes strongly and positively enhanced (image on right) in
comparison to the T1 weighted image acquired before injection (left).61

3.2.2 Superparamagnetic contrast agents—Superparamagnetic contrast agents are

colloidal materials made up of particles (~ 5-200 nm in diameter) in suspension. These
particles are composed of small crystallites (1-10 nm) that contain thousands of magnetic
ions, usually iron, that are randomly oriented. In the presence of an external magnetic field,
the crystallites align with the field, leading to a superspin which renders the material
magnetic. The total spin of the particle is much larger than the sum of the individual metal
ion spins which can result in a very high relaxivity. In the absence of the applied field, the
material is no longer magnetic. The crystallites are made of a core of nonstoichiometric
metal (usually iron) oxides covered by a coating, such as dextran, citrate, oleate, or other
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nonimmunogenic polymers in order to avoid aggregation and reduce their toxicity.62–66 The
first superparamagnetic contrast agents had very high r2/r1 ratios and predominantly affected
T2. As a result they were referred to as T2-agents or negative contrast agents because they
provide darkened MR images. However there are many examples of superparamagnetic iron
oxide particles that also have a large r1 and can serve as effective T1 or T2 contrast agents.

Superparamagnetic iron oxides can be classified in three groups according to the size of the
particulate: (1) Ultra-Small Superparamagnetic Iron Oxide (USPIO) particles with a
diameter of d < 50 nm; (2) Small Superparamagnetic Iron Oxide (SPIO) particles with a
diameter of 1 μm > d > 50 nm; and (3) Micron-sized Particles of Iron Oxide (MPIO) with a
diameter in excess of a micron.67 Iron oxides that have been approved for clinical use or that
have undergone human clinical trials are listed in Table 2 and 3 above. These agents are also
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referred to generically as Superparamagnetic Iron Oxide Nanoparticles (SPION).

Figure 8 shows the effect of a T2 agent on tissue relaxation times. Although the contrast
agent shortens T1, the effect on T2 is very large. The large magnetic moment of
superparamagnetic relaxation agents also generates a local magnetic inhomogeneity and
shortens the T2* of tissue which creates signal loss on gradient echo T2* weighted images.

Exemplary images from a patient with primary central nervous system (CNS) lymphoma are
shown in Figure 9, before and 24 hours after intravenous injection of ferumoxytol.68 T2-
weighted images obtained before ferumoxytol show regions of hyperintensity due to tumor
associated edema. After 24 hours, ferumoxytol has accumulated in the tumors and several
areas of deep white matter lesions are enhanced through strong signal loss due to
ferumoxytol uptake in these regions.
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3.2.3 Chemical Exchange Saturation Transfer (CEST) Agents—CEST agents are

molecules that possess exchangeable protons, such as NH, OH, or exchangeable water
molecules, that resonate at a chemical shift that is different from the bulk water signal. In a
CEST experiment, the exchangeable protons of the CEST agent are irradiated with an off-
resonance saturation pulse. Their magnetization (i.e. spin polarization) is transferred to the
protons of the bulk water pool by exchange between both pools (saturated proton pool and

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bulk water proton pool), which leads to a reduced intensity of the water signal. This in turn
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generates MRI contrast.

For a CEST agent to be effective there needs to be good frequency separation between the
exchangeable protons and the bulk water resonances, especially since the bulk water
resonance is broadened in vivo because of local magnetic field inhomogeneities.69 To
overcome this problem, the chemical shift difference needs to be maximized. This can be
achieved by either increasing the magnetic field strength (higher field strengths for the MR
experiment) or by using paramagnetic CEST (ParaCEST) agents. Typically, ParaCEST
agents contain paramagnetic ions to shift the signal of the labile proton tens to hundreds of
ppm away from the bulk water resonance.

Some CEST contrast agents are known molecules that are already approved for intravenous
administration. Examples include glucose (exchangeable OH) and the X-ray contrast agent
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Iopamidol (exchangeable NH) (Figure 10), that are currently being used in human clinical
trials.70–72

3.2.4 Direct detection agents—MRI is an insensitive technique. To overcome this low

sensitivity clinical MRI detects hydrogen, the most sensitive NMR nucleus, and primarily
detects water because of its very high (10s of molar) concentration in tissue. Other proton
containing molecules or heteronuclei can be detected, such as 13C, 23Na, 31P, and 19F, but
very high concentrations are usually required. Nonetheless there are numerous examples of
direct detection agents. The T1-, T2-, and CEST agents described above are contrast agents
that change the local MR water signal and create image contrast. These agents are not
directly detected, but are seen via their effect on the bulk water signal. Direct detection
agents do not generate contrast and in general have little to no background signal, but do
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suffer from poor sensitivity.

To overcome the sensitivity problem nanoparticle containing perfluorocarbon (PFCs)

emulsions, providing huge payloads of 19F atoms, have been used for direct detection in 19F
MRI.73–74 These perfluorocarbons are the most biologically inert organic xenobiotics and
usually, even at high doses, their toxicity is very low.75 Selected examples of PCFs are
illustrated in Figure 11.

To overcome the low water solubility of traditional PFCs, a new generation of hydrophilic
fluorinated molecules has emerged. Annapragada and coworkers developed stable liposomal
formulations of hydrophilic fluorinated molecules that allow simultaneous imaging of
multiple targets (Figure 12).76
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The natural abundance of 19F (100%), its spin (1/2), its high gyromagnetic ratio (40.08
MHz/T, 1H: 42.58 MHz/T), and sensitivity (83% of 1H) makes 19F a promising candidate for
direct detection imaging. In addition, the chemical shifts of 19F containing compounds vary
over a broad range (>350 ppm) and the in vivo background signal is very low due to the
natural abundance of the element in the human body. In addition, the concentration of agent
can be measured directly from the intensity of the signal. While not widely available, it is

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possible to modify MRI hardware to directly detect both 1H and 19F, and in this way the
visualization of the 19F agent can be localized to the anatomic 1H (water) image.
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It is also possible to directly detect the 1H resonance of a direct detect agent, but because of
the narrow chemical shift range of 1H, there is usually a significant background signal from
metabolites. A further limitation of direct detection agents is the long T1 of the nucleus
being detected. After excitation one has to wait until the T1 magnetization is sufficiently
recovered before applying a second excitation pulse. The acquisition time window in vivo is
limited because the agent may be washing out of tissue with time, and there is a practical
limit of keeping the subject in the scanner. If the T1 was very short, then excitation pulses
could be acquired continuously allowing for signal averaging. Aime et al. showed over 20
years ago that a paramagnetic complex exhibiting a large chemical shift could overcome
these problems to some extent.77 Yb-DOTMA has 4 equivalent CH3 groups (12 H per
molecule) that are well shifted outside the diamagnetic window. Moreover the Yb(III) ion
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causes efficient T1 relaxation of the CH3 protons but does not result in severe
linebroadening. The chemical shift of the CH3 group is very sensitive to temperature and
several lanthanide complexes of DOTMA have been used in MR thermometry applications.
78–79 Yang et al. took a different approach and encapsulated sodium 3-(trimethylsilyl)-1-

propanesulfonate (DSS) at a high concentration inside liposomes.80 DSS has a zero ppm
chemical shift, outside the range of metabolites. A small amount of Gd-HPDO3A was
included to shorten the T1 of the DSS protons. They showed that liposomes could be directly
detected at concentrations as low as 50 pM.

Parker and coworkers used a rational, systematic approach to design what they termed
ParaSHIFT agents (Figure 13). They first designed compounds with a large number of
equivalent fluorine atoms and incorporated a lanthanide ion at an optimal distance to the
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fluorine to optimize T1 shortening. In this way, they showed that the sensitivity could be
increased by a factor of 25 compared to the diamagnetic compound. More recently they have
extended this approach to lanthanide complexes with a shifted proton signal (e.g. from a t-
butyl group with 9 equivalent protons).81–82

The insensitivity of NMR arises from the low energy difference between the ground and
excited state which results in a large population of spins in the excited state at thermal
equilibrium. If the nuclear spins could be polarized far beyond thermal equilibrium
conditions, then the sensitivity could be increased by several orders of magnitude. The
polarization of spins is enhanced by a non-equilibrium distribution of nuclear spins called
the hyperpolarized state.85 This technique requires nuclei with long T1 values such that
relaxation does not occur in the time the molecule is hyperpolarized to when the image is
acquired, and includes nuclei such as 3He, 129Xe, 13C, 15N, 6Li.86
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Initially hyperpolarization was applied to gases like 3He and 129Xe which have very long T1
values. These gases are used clinically at a number of sites to image the lung architecture
after inhalation of the gas.87–90 In the last decade 13C hyperpolarization has been widely
employed and there is now a commercial apparatus available to hyperpolarized 13C for
human applications. For example, hyperpolarized [1-13C] pyruvate was used by Nelson et al.
in patients with prostate cancer.9 Pyruvate is metabolized to lactate, alanine, and bicarbonate

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 Wahsner et al. Page 13

in a matter of seconds, and each of these can be visualized and quantified because of the
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chemical shift differences between these metabolites. The ratio of pyruvate metabolites can
be used to grade tumor aggressiveness and monitor treatment response.

The most widely used method for hyperpolarization is dynamic nuclear polarization (DNP).
91 In this method, a stable organic radical, usually trityl, is frozen along with the compound
to be hyperpolarized. This sample is placed inside a DNP polarizer, comprising a
superconducting magnet (B0 = 3 to 5 T) and a liquid helium cooled sample space
(maintained cold at 1 to 5 K). Under these conditions the radical’s unpaired electrons are
aligned with the external magnetic field, which leads to a hyperpolarized state, and this
electron hyperpolarization is transferred to the 13C nucleus using microwave radiation close
to the MR frequency of the electron spin. Before the hyperpolarized sample can be used in
vivo, it needs to be rapidly dissolved in an appropriate solvent, heated to physiological
temperature, and separated from the radical. Generally a superheated solvent at 180 ºC and
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10 bar is used.92

Another frequently used method for polarization is the parahydrogen-induced polarization

(PHIP)93 that induces hyperpolarization through the reaction of parahydrogen (its two
proton spins are aligned antiparallel) with an unsaturated substrate containing double or
triple bonds.91 Typically, parahydrogen is introduced through catalytic hydrogenation to the
unsaturated compound. Subsequently, the nuclear polarization of a vicinal 13C nucleus is
achieved by converting the nonequilibrium spin polarization of parahydrogen by diabatic-
adiabatic magnetic field cycling or by radiofrequency pulses.94

A third and related approach is signal amplification by reversible exchange (SABRE).

SABRE involves the transient coordination of a substrate molecule (usually containing a
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coordinating atom like a pyridyl N) to an organometallic catalyst. Parahydrogen (H2) also

coordinates the catalyst and then transfers its polarization to the substrate in a catalytic
manner. Unlike in PHIP, the substrate molecule is unchanged.95–96

3.3 Biophysics of magnetic resonance imaging

There are many ways to achieve contrast in a MR image without giving an exogenous
contrast agent.3, 97–99 Chemical shift difference can provide contrast between mobile lipids
and water. The density of water protons in tissue also provides contrast (low in lung and
bone, higher in soft tissue and fluids). There are a number of contrast mechanisms that
exploit differences in the properties of water in different tissues. For instance the relaxation
times T1, T2, T1ρ (T1 in the rotating frame) can be very different in adjacent tissues and
images can be acquired using pulse sequences that are weighted to provide more (or less)
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signal intensity for a short relaxation time. Contrast can also be generated from
physicochemical properties of water such as diffusion and flow. Magnetization transfer from
the solid-like macromolecules to bulk water is another mechanism, as is saturation transfer
(discussed in CEST contrast). Exogenous contrast agents can further exploit these
mechanisms by amplifying the effect. The majority of contrast agents serve to shorten the
relaxation times of water, while CEST agents exploit a saturation transfer mechanism. In this
section we describe the mechanisms by which contrast agents affect relaxation times or
saturation transfer.

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3.3.1 Relaxation agents—Relaxation agents are primarily described by their

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“relaxivity” (r1 or r2), that is defined as the extent to which the contrast agent can modify the
relaxation rate of tissue water (1/T1 or 1/T2) The conventional units for relaxivity are mM−1s
−1 (per millimolar per second, and sometimes L·mol−1s−1). Sometimes, the relaxation rate

enhancement of the contrast agent is referred to as 1/Tip (i = 1,2) as the paramagnetic

contribution to the relaxation rate.

Figure 14 shows the effect of a typical ECF agent on the relaxation rate (a) and on the
relaxation time (b) of three different tissues: heart (T10 = 1200 ms), liver (T10 = 590 ms),
and subcutaneous fat (T10 = 340 ms).100 Panel (a) shows that increasing the contrast agent
concentration increases the tissue relaxation rate in the same linear fashion for all three
tissues, although the intercept differs among the tissue types. The slope of the lines in panel
(a) is the relaxivity, r1. Panel (b) shows the same data expressed as relaxation times. At lower
concentrations (left part of the graph), the amount of contrast agent has a higher effect on
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tissue with longer T1, whereas at higher concentrations of contrast agent (right part of the
graph), all tissues approach a similar relaxation time. Thus the detection sensitivity of the
contrast agent will be greater in tissues with long relaxation times.

The relaxation rate of the tissue (1/T10) varies with tissue type, usually in the range 0.5 – 2 s
−1 for most tissues and tumors, and increases with magnetic field strength.60, 101 In

designing experiments and new contrast agents it is important to consider both the relaxivity
of the agent and the inherent relaxation rate of the tissue, since both are involved in
determining the signal change observed. Figure 15 illustrates this point using relaxivity data
for Gd-DOTA59 and MS-32515 measured in human plasma and compared to brain grey
matter (GM) and white matter (WM) T1 values measured at 1.5 and 4.7 T.60 The relaxation
times of GM and WM increase by about 50% with increasing field while Gd-DOTA
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relaxivity only decreases by about 10%. As a result, the percent change in brain tissue
relaxation rate that one would observe for a given Gd-DOTA concentration is actually higher
at 4.7 T, despite its lower relaxivity at this field strength. Even for MS-325 where the
relaxivity at 1.5 T is more than double that at 4.7 T, the change in brain tissue relaxation rate
for a 0.3 mM MS-325 concentration is only about 40% higher at 1.5 T.

In the case of transverse relaxation, the T2 of tissues is generally very short and usually
decreases with increasing field strength. Thus much higher concentrations of contrast agent
and/or much higher relaxivities are required to measurably affect T2.102

T1-contrast agents: Brownian motion of a paramagnetic complex or particle generates a

fluctuating magnetic field that induces relaxation in nearby water molecules. In order to
transmit the relaxation effect to the bulk, the exchange between the water interacting with
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the metal ion and the bulk needs to be fast (>106 s−1). Paramagnetic induced T1 relaxation
directly depends on the spin quantum number (as a function of S(S+1)) and inversely on the
distance (rMH) between the metal ion and the proton of the water as a function of 1/rMH6.
Gadolinium(III) with S = 7/2 and high spin manganese(II) or iron(III) with S = 5/2 have
been the most widely used as contrast agents. The shortest rMH distance can be achieved
through a direct bond between the metal ion and the water molecule, thus occupying a
coordination position in the inner-coordination sphere of the metal ion. The exchange rate of

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 Wahsner et al. Page 15

this water molecule must be fast (> 106 s−1) in order to maximize the transfer of the
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relaxation effect to the bulk water. The absence of ligand field stabilization energy of Gd(III)
and Mn(II) makes their complexes quite labile, thus favoring the fast chemical exchange of
the bound water molecule. These ions also have a symmetric S electronic ground state which
results in electronic relaxation typically in the nanosecond timescale, and this allows an
efficient nuclear T1 relaxation.

From a chemical standpoint, three contributions to relaxivity can be considered:103

• Inner-sphere relaxation where a water ligand directly bound to the metal is

relaxed and transmits the relaxation effect to the bulk water through exchange
with another water molecule.

• Second-sphere relaxation where hydrogen bonded water molecules in the second

coordination sphere, or exchangeable hydrogen atoms (such as O-H, N-H)
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undergo relaxation and exchange.

• Outer-sphere relaxation, where water molecules that diffuse close to the

paramagnetic compound can also be relaxed.

Because of the difficulty to experimentally distinguish water molecules in the second-sphere

from outer-sphere water, these two groups are often referred to as outer-sphere water
molecules. Relaxivity can be factored into these two components of inner-sphere r1IS and
outer-sphere (r1OS relaxivity (equation 2):

r1 = r1IS + rOS
1 (2)
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It should be noted that r1OS can represent a major component of the observed relaxivity and
is about 40% of the relaxivity of approved ECF agents.

Inner-sphere relaxivity: For water ligands in the inner-sphere, relaxivity depends on the
extrinsic parameters applied magnetic field strength and temperature, and on intrinsic
molecular factors including: the number of water molecules in the inner-coordination sphere,
q; the kinetics of water exchange kex = 1/τm where τm is the mean residency time of the
water ligand; the rotational dynamics of the molecule, described by a rotational correlation
time τR; the electron spin S of the complex; and the electronic relaxation times T1e and T2e,
which are sometimes referred to as τS. Figure 16 summarizes these molecular factors along
with outer-sphere relaxation that is described by the translational diffusion correlation time
τD and a distance of closest approach a (Figure 16).
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Combining the definition of relaxivity with the Bloch equations for a system in fast two site
exchange, where one pool (bulk water) is present at much higher concentration than the
other pool (metal bound water), one arrives at equations 3 and 4 which describe inner-sphere
relaxivity. Here Tim is the Ti (i = 1 or 2) of the coordinated water molecule and is described
by the Solomon-Bloembergen-Morgan equations of paramagnetic relaxation theory,104–108
equations 5 and 6. Here rMH is the metal ion to water hydrogen distance, γH is the proton
magnetogyric ratio, g is the electronic g factor, μB is the Bohr magneton, µ0 is the vacuum

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permeability, and ωS and ωH are the Larmor frequencies of the electron and proton,
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respectively. There is also a correlation time for the magnetic fluctuation (τc) that is the
shorter of the rotational correlation time (τR), the electronic relaxation time (T1e or T2e), or
the water residency time (τm) defined by equation 7. The T1 effect is dipolar and 1/T1m
depends on the electronic spin state of the complex, indicating that larger S values give
greater relaxation. It depends on both the nuclear and electron magnetogyric ratios
indicating that relaxation is greater for H than for other nuclei with lower γ values.109 The
T2 effect is contributed by both dipolar (through space) and scalar (contact, through bonds)
interactions. The scalar contribution depends on the hyperfine coupling constant between the
metal ion and the water proton and a correlation time τsc for that interaction, equation 8. The
scalar contribution to proton relaxation is small for Gd(III) complexes but can be appreciable
for Mn(II) and Fe(III).

q/[H 2O]
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r1IS = (3)
T 1m + τm

−1 −1 −1 2
q 1 T 2m τm + T 2m + Δωm
r2IS = (4)
[H 2O] τm T −1 −1 2
+ Δω2m
2m + τm

2 2 2
1 2 μ0 γ H ge μBS S + 1 7τc2 3τc1
= + (5)
T 1m 15 4π 6
r MH 1 + ωSτc2 1 + ω2H τ2c1
2 2
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2 2 2
1 1 μ0 γ H ge μBS S + 1 13τc2 3τc1 S S+1 A 2
= 4τ c1 + + + τ (6)
T 2m 15 4π r6MH 1 + ω2Sτ2c2 1 + ω2H τ2c1 3 ℏ sc

1 1 1 1
= + + ; i = 1, 2 (7)
τci τ R τm T ie
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1 1 1
= + (8)
τsc τm T 1e

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3τC
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1 C
= 6 ⋅ (9)
T 1m r 1 + ω2H τ2C
GdH

r1IS = C ⋅ q ⋅ τr (10)

Because the electron Larmor frequency is much higher than the proton frequency, the term
ωsτc2 becomes very large by about 0.1 T and thus the “7” term in equation 5 becomes
negligible at applied fields above 0.1 T and reduces to equation 9 where C represents the
physical constants in equation 5. For simple metal complexes, T1m is always large (several
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microseconds) compared with the mean residency time of the metal-bound water molecule
(submicrosecond at 37 °C for most Gd(III) and Mn(II) complexes, so T1m >>τm). For such
complexes, the dominant correlation time at most imaging fields (B0 > 0.2T) will be the
rotational correlation time. In these cases, inner-sphere relaxivity can be approximated to
equation 10 and depends only q and the rotational correlation time.

In these complexes, the relaxivity is almost always limited by the fast rotation of the
complex (short τR, ~ 50 – 200 ps at 37 ºC).110–111 The relaxivity could be increased by
slowing down its rotation (longer τR) by enhancing the molecular weight of the
paramagnetic compound. For instance, this can be accomplished by immobilizing the
complex, covalently or non-covalently, on a macromolecule.

The fluctuating field can also be caused by the water molecules that are in contact with the
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metal complex, but this is only observed when the rotational correlation time is very long
and water exchange is extremely fast.112

Electronic relaxation: At very low fields (< 0.1 T), electronic relaxation is the dominant
correlation time for Gd(III) and Mn(II) complexes. The influence of the electronic relaxation
on the relaxivity of paramagnetic complexes depends mainly on the decay of the electron
spin magnetization in the direction parallel to the external magnetic field (T1e). This decay is
too fast in order to be directly measured, but the transverse electronic relaxation (T2e) can be
estimated by EPR. For high spin ions like Gd(III) and Mn(II), the main relaxation
mechanism is a transient zero-field splitting (ZFS). ZFS describes interelectronic
interactions in paramagnetic compounds with two or more unpaired electrons. The
modulation of the zero-field splitting depends on the perturbation of the ligand field through
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rotation, vibration and other motions. This splitting is the result of inter-electronic repulsion,
spin-orbit coupling and the action of the ligand field on the unpaired electrons of the
paramagnetic metal center. The spin Hamiltonian formalism can be written as follows
(equation 11):113

2 1 2 2 2
H ZFS = S · D · S = D S z − S + E S + + S − (11)
3

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where D and E describe the magnitude of the operator D parallel and perpendicular to the z
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axis. This perturbation lifts the 2S+1 degeneracy at zero field, giving S + 1/2 (for half-
integer S) doubly degenerated zero-field spin states or S + 1 (for integer S) zero-field spin
states where one of them is non-degenerated (since mS = 0), that can be written as linear
combinations of the Zeeman mS states. If an external magnetic field (B0) is applied, the
doubly degenerated levels will be lifted. In the case of high spin ions in a symmetrical
environment, such as the aqua ions of Cr(III), Fe(III), Mn(II), Gd(III) or Eu(II), the zero-
field splitting is averaged out. An extension of this reasoning can be applied for Gd(III) and
Eu(II) complexes with polyaminocarboxylates, where f electrons are mostly shielded from
the ligand field and result in a small, but non-zero ZFS.

Although Gd(III) and Mn(II) complexes have non-zero ZFS, the effect of this static ZFS can
usually be ignored at most field strengths where the Zeeman energy is much larger than the
ZFS energy. However NMRD relaxometers can operate at very low proton Larmor
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frequencies, e.g. 10 kHz, and there the Zeeman energy may be lower than the ZFS. In such
instances the widely used SBM relaxation theory does not apply.114–117 As alternatives to
Gd(III) are increasingly proposed it is important to consider the magnitude of the ZFS of
those complexes. If the ZFS is much larger than the Zeeman energy, then SBM theory may
no longer apply.

In addition to this static ZFS, solvent collisions can induce distortions in the molecule
resulting in a transient ZFS, and this transient ZFS can serve as an efficient relaxation
mechanism. The transient ZFS broadens the line in EPR spectra.118 The electronic
relaxation rates are given by equations 12 and 13,119 where τv is a correlation time that
describes the modulation of the transient ZFS, and Δ2 is the square of the trace of the ZFS
tensor.
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1 12 1 4
= Δ2τv + (12)
T 1e 5 1 + ωSτv 1 + 4ω2Sτ2v
2 2

1 12 5 2
= Δ2τv 3 + + (13)
T 2e 10 1 + ωSτv 1 + 4ω2Sτ2v
2 2

Thus 1/T1e decreases with the square of increasing applied field. As the field is increased,
eventually 1/T1e < 1/τR and rotation dominates the overall correlation time. For instance, at
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0.02 MHz, the relaxivity of Gd-DOTA is r10.02MHz = 11.3 mM−1s−1 compared to 7.7 mM−1s
−1 for Gd-DTPA at 25 °C, owing to the much longer T of Gd-DOTA. But, at 20 MHz, the
1e
relaxivity of these two complexes is almost identical: r120MHz = 4.7 mM−1s−1 for Gd-DOTA
and 4.8 mM−1s−1 for Gd-DTPA.120 Complexes with higher symmetry will have smaller ZFS
energies and thus, longer electronic relaxation times. For example, the axial ZFS parameter
D is 24 mT for Gd-DOTA but 56 mT for Gd-DTPA.107. Intramolecular electronic relaxation
between several Gd(III) ions can shorten T1e if the metals are very close ( << 10 Å).121–122

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However this effect is only manifest on relaxivity at low fields. In the design of novel
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optimized MRI contrast agents, electronic relaxation is usually not taken into account, since
its contribution to relaxivity is negligible at the magnetic field strengths that are typically
used in clinic (≥1.5 T).

The metal-water hydrogen distance (rMH): Because this distance enters as the inverse
sixth power to the relaxation rate, small changes in rMH could have a profound effect on
relaxivity, especially if this distance could be tuned. Caravan, Raitsimring and coworkers
showed that the Gd-H(water) distance could be determined on frozen solutions using high
field proton electron nuclear double resonance (1H ENDOR) spectroscopy.121, 123–128 They
investigated a range of q=1 and q=2 complexes with different water exchange kinetics and
found that rGdH was consistently 3.05 Å distributed within a range of ±0.07 Å. That is, there
is a normal distribution of Gd-H distances ranging from 2.98 – 3.12 Å with the average
distance being 3.05 Å (note that the initial paper gave a distance of 3.1Å obtained at a lower
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field; this was refined by measurements at very high field).121, 127 They compared the DTPA
derivatives Gd-DTPA, Gd-BOPTA, and MS-325 and found that the ENDOR spectra were
superimposable indicating a common rGdH, although earlier reports using indirect measures
of rGdH had proposed differences in rGdH among these complexes.129–130 They measured
Gd-DOTA, Gd-DOTMA, Gd-HPDO3A, and two Gd-DOTAla derivatives and again found
rGdH = 3.05 Å and no difference in the ENDOR spectra. They also measured 3 different q=2
complexes, and there too observed the same rGdH value, see Figure 17.

Number of water molecules (q): Inner-sphere relaxivity is directly proportional to the

number of water ligands (see equations 3 and 4). Increasing the hydration number (q) should
increase the inner-sphere relaxivity, but an increase in q is often accomplished at the cost of
decreased thermodynamic stability and/or kinetic inertness.131 A second consideration is
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that two cis coordinated water ligands can often be displaced by a coordinating anion in
vivo. Thus it is important to know the hydration state of the complex in solution. Unlike
certain other metal ions, the coordination number of Gd(III) and high spin Mn(II) varies and
may not be easily predicted. Gd(III) complexes in aqueous solution are often CN9 but many
CN8 and even CN7 and CN10 examples exist.132–133 Mn(II) complexes in aqueous solution
tend to be CN6 and CN7.

There are several physical methods that allow estimation of the number of water ligands
coordinated to Gd(III). There are surrogate approaches where Gd(III) is replaced by another
Ln(III) similar in ionic radius. For instance the induced paramagnetic shift of H217O is
proportional to q under fast exchange conditions. Geraldes and Peters and coworkers showed
that the H217O shift induced by the Dy(III) complex could be measured and compared to
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[Dy(H2O)8]3+ to estimate q for the new complex.134 However, this method requires large
amounts of sample (>10 mM).

Another method is based on luminescence lifetime measurements of the corresponding

Eu(III) or Tb(III) analogs measured in both H2O and D2O. Coordinated H2O quenches the
emission of Eu(III) or Tb(III) more efficiently than D2O. The reason for this lies in the
different energy levels of the vibrational overtones of the O-H and O-D oscillators. The
vibrational overtones of O-D are lower in energy in comparison to an O-H oscillator, which

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makes the energy transfer from Eu(III) or Tb(III) excited states into the O-D oscillators
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higher harmonics less likely. Horrocks and Sudnick showed that a simple empirical equation
could be used to relate these decay rate constants to q,135 and some refinements on this
approach have been reported.136–137 A benefit of this approach is that low µM
concentrations can be used and the method is also generally useful for estimating q in
complex matrices such as biological samples.

Another more direct method is electron-nuclear double resonance (ENDOR) spectroscopy of

the Gd(III) complex.126 Raitsimring et al. used high field pulsed 17O ENDOR and showed
that the presence of coordinated water ligands was associated with two narrow lines with a
splitting of about 1.33 MHz originating from the −1/2 ⇔ +1/2 transitions of the 17O nuclei.
125 By comparison of the amplitude of these transitions to the Gd(III) aqua ion (q = 8), the

number of coordinated waters in the unknown complex could be determined.

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As noted above, the Gd-H(water) distance determined by 1H ENDOR was invariable, and
about 3.05 Å. As a result the position of the ENDOR transition associated with this
coordinated water was constant. Raitsimring et al. exploited this effect to estimate q by 1H
ENDOR. The difference in the intensity of the 1H ENDOR spectra measured in H2O and in
D2O is proportional to the number of exchangeable protons. This technique allows the direct
utilization of the gadolinium(III) compound and also protein bound Gd(III)-complexes can
be directly measured.124, 126

Theoretical calculations can also provide an idea of the hydration number of the Gd(III),
however it should be cautioned that the energy difference between CN9 and CN8 Gd(III)
complexes is small and can also be strongly influenced by the presence of solvent water. X-
Ray crystallography provides evidence of the hydration number in the solid state, but again,
this may not reflect the hydration number in solution.109 Furthermore weakly coordinating
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ions such as nitrate or acetate may crystallize coordinated to a Gd(III) complex, but these
will typically be displaced by water ligands in aqueous solution.

Gale et al. developed a simple NMR method to measure the hydration number of Mn(II)
complexes.138 They demonstrated that the maximum in a plot of transverse O-17 relaxivity
(r2O) versus temperature was directly proportional to the number of coordinated water
ligands. This technique could be performed with micromolar concentrations of chelate,
allowing for its use in interrogating the hydration number of protein-bound chelates. From
the same data set, one can also determine the water exchange kinetics.

The tumbling of the molecule (τR): As discussed above, at the most common field
strengths used for imaging, the dominant correlation time is almost always rotational
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diffusion. For instance, for small monomeric gadolinium(III) complexes, τR is about 0.1 ns
and at low magnetic field strength and 37 ºC, slowing down the rotation will result in an
increase of the relaxivity. Reducing the rate of molecular tumbling (that is, making τR
longer) will enhance the relaxivity of a T1 contrast agent at low field strength. However at
higher fields, relaxivity will increase with increasing τR up to a point, after which relaxivity
will decrease with further increases in τR. This is because of the 3τc/(1+ωH2τc2) term in eq
5; as τc increases, relaxivity will increase until the product ωH2τc2 > 1 which will occur at

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high fields and/or high values of τc. This is illustrated in Figure 18 where field-dependent
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relaxivity profiles, so called nuclear magnetic relaxation dispersion (NMRD), are shown for
Gd(III) complexes with increasing rotational correlation times. Figure 18A shows the
NMRD profile for MS-325 in phosphate buffered saline where τR is on the order of 0.1 ns.
In the presence of human serum albumin (Figure 18C), much of the MS-325 is protein
bound with an effective τR of about 10 ns.17–18, 139–141 Slow rotation results in a multifold
increase in relaxivity at low fields, but at 7 tesla the relaxivity has dispersed to the point that
the unbound form of MS-325 has slightly higher relaxivity. Figure 18B shows the per
Gd(III) NMRD profile of EP-1084 which is a contrast agent comprising 4 Gd-DTPA
chelates and two fibrin binding peptides.142 This compound has a relatively high molecular
weight resulting in an intermediate τR. While the relaxivity of EP-1084 is lower than
MS-325/HSA at low fields, by 1.5 T the relaxivity is almost the same and at 3 T and 7T the
relaxivity of EP-1084 is higher than MS-325/HSA. All three examples involved a Gd-DTPA
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derivative where the differences in relaxivity arose solely because of differences in rotational
correlation time. Strategies to optimize the rotational correlation time are further discussed
in section: 4.2 High relaxivity Gd(III)-contrast agents: 4.2.2. Optimizing the rotational
correlation time (τR) of Gd(III)-based contrast agents.

Rotational diffusion can be estimated through several physical methods, such as NMR
relaxation, fluorescence lifetime measurements, or can be calculated from the Stokes-
Einstein equation.2

The exchange rate of the water molecule: The water exchange rate kex (1/τm where τm is
the residency time of the coordinated water) is another key and tunable molecular parameter.
Relaxivity has a 1/(T1m + τm) dependence. For small Gd(III) and Mn(II) complexes, T1m >>
τm, and so these complexes tend not to be limited by slow water exchange. However if
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rotation is slowed and T1m is decreased, then water exchange may limit relaxivity. For other
metal ions, water exchange may be slow relative to T1m.143

Figure 19 shows a simulation of the effect of the water exchange on gadolinium(III)

complexes at three different magnetic fields: 0.47, 3.0, and 9.4 T. At each of these field
strengths, the rotational correlation time (τR) is near the optimum value for that specific field
strength, that is 20, 1.5 and 0.5 ns, respectively.144 Note that the y-axis range differs because
the theoretical maximum relaxivity decreases with increasing field strength. At 0.47 T, very
high relaxivities can be attained, but only for a narrow range of water exchange rates. At the
higher fields a broader range of water exchange rates can yield near optimal relaxivity.

Relaxivity is limited by both very slow (long τm) and very fast exchange (Figure 19). In the
latter case, if exchange is too fast then water is not present long enough for there to be a high
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probability of relaxation. Modifications of τm are usually related to the stabilization or

destabilization of intermediates that participate in the water exchange mechanism and/or
with changes in the population of the isomers present in solution. For instance, for DOTA-
type ligands, it was found that water exchange is around 50 times faster in the twisted-square
antiprismatic (TSAP) isomer compared to the square antiprismatic (SAP) isomer. The main
explanation for this is the steric crowding at the water binding site, which favors the release
of the metal-bound water in a dissociative mechanism.145 Figure 20 shows a simplified

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 Wahsner et al. Page 22

vision of the SAP and TSAP conformations in a DOTA-type complex. From a geometric
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point of view, these two isomers can be distinguished by the twist angle between the plane
defined by the chelating oxygen atoms and the chelating nitrogen atoms: ~ 25 °for the TSAP
isomer and ~40 ° in the case of the SAP form.

For DOTA-like structures, two sources of chirality should be considered: the orientation of
the chelates (δδδδ or λλλλ) and the helicity of the pendant arms [Δ (clockwise
arrangement) or Λ (counter clockwise arrangement)] (Figure 21).146–148

The TSAP isomer has the same stereochemistry at both positions [Δ(δδδδ) or Λ(λλλλ)],
whereas the SAP isomer has the opposite stereochemistry at the two positions [Δ(λλλλ) or
Λ(δδδδ)]. Interconversion between the two isomers is possible by ring inversion (δδδδ ↔
λλλλ) or by arm rotation (Δ ↔ Λ). Both, arm rotation and ring inversion performed in a
successive manner or concerted way lead to an enantiomerization process. Because of steric
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repulsion between the nitrogen and oxygen donors, the distance between the N4 and O4
plane is increased in the TSAP isomer,149 thereby making the metal center less accessible
for the coordination of a water molecule.145 On the other hand, within this isomer more
bulky pendant arms can be accommodated. Substitution on the α position of the pendant
arms slows or completely eliminates the arm rotation process,150–153 while substitution on
the cyclen backbone restricts the ring inversion motion.154–157 Substitution at both positions
effectively locks the macrocycle into a single conformation.158–161

The water exchange rate can be measured from the temperature dependence of the 17O
NMR transverse relaxation rate of solvent water in the presence and absence of the metal
complex at high fields.143

Outer-sphere relaxivity: In addition to the inner-sphere contribution to relaxivity, the

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second-sphere and in general, the outer-sphere contribution also needs to be taken into
account. The relaxivity derived from the water molecules diffusing close to the
gadolinium(III) complex can be predicted from the hard-sphere model of Hwang and Freed,
162 where the relaxation depends primarily on the diffusion coefficient of water and the

distance of closest approach to the metallic center.

Second-sphere relaxivity is an operational definition that refers to complexes that have water
molecules or exchangeable protons in the second coordination sphere that have a residency
time longer than the diffusion lifetime. Second-sphere relaxivity is described by the same
equations as inner-sphere relaxivity but here the dominant correlation time is the residency
time of the second-sphere protons. In general, this effect is seen when independently
determined molecular parameters cannot account for the observed relaxivity.109 Figure 22
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shows some selected examples of q = 0 complexes with an important second sphere effect.

For instance, the relaxivities of [Gd(TTHA-P)]3– in buffer and in the presence of HSA are
2.1 mM−1s−1 and 8.0 mM−1s−1, respectively and the relaxivities of [Gd(DO3A-pic-bip)]2– in
buffer and in the presence of HSA are 3.1 mM−1s−1 and 7.0 mM−1s−1, respectively.
Although these values are lower than that of q = 1 compounds, their relaxivity is non-
negligible. When bound to human serum albumin, the relaxivity is increased by 3 to 4-fold

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 Wahsner et al. Page 23

due to the long lived proton(s) near the Gd(III)-center.17 [Gd(C11-DOTP)]5− is also a q = 0
complex but its relaxivity is much higher than that of [Gd(TTHA-P)]3−, that is a molecule of
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similar size. The reason for this is the negatively charged surface due to the phosphonate
moieties, which also furnish a favorable arrangement of the water molecules in the second
coordination sphere.

T2-contrast agents: In the case of T2-contrast agents, as T2 is shortened, the water

linewidth increases, so the signal decreases, leading to a negative image contrast. At the
molecular level, the rotation of the complex in solution creates a fluctuating magnetic field
that gives rise to T2 and can be described according to the Solomon-Bloembergen-Morgan
(SBM) theory, equation 6.2 Pure T2 relaxation requires access of the water molecule to the
contrast agent. For discrete complexes SBM theory can also describe T2 relaxation. However
for 1/T2m, there is a field independent term for both dipolar and scalar relaxation such that r2
does not decrease at high fields the way that r1 does. In addition there is an additional
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relaxation mechanism called Curie spin relaxation that results in increased r2 at high fields,
especially for ions with large magnetic moments.163

T2* takes into account the inhomogeneity of the local magnetic field (B0) and is related to
T2 by equation 14:

1/T2* = 1/T2 + γΔB0 (14)

The magnetic susceptibility of the paramagnetic agent causes local changes in B0 and
induces this T2* effect. This magnetic susceptibility depends on the concentration of the
contrast agent and its molar susceptibility. Since Tb(III) and Dy(III) have larger moments
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than Gd(III), their complexes could be used as contrast agents with increased susceptibility.
164 These effects can be pronounced in vivo where compartmentalization of the contrast

agent naturally occurs. For instance as the contrast agent passes through the blood vessels in
the brain there is a large susceptibility gradient between the blood, where the contrast agent
resides, and the brain tissue where the contrast agent is absent. This creates a large T2*
relaxation effect and is used clinically to measure brain perfusion.165

Iron oxide nanoparticles have much larger magnetic susceptibilities than discrete
coordination complexes. SPIONs are all T2-contrast agents, that de-phase the spins of the
nearby protons of the water molecules, leading to a decrease in the signal. In the case of
superparamagnetic particles, outer-sphere theory for T2 relaxivity predicts that their
effectiveness is highly dependent on both the saturation magnetization (MS) value and the
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effective radius (r) (equation 15):

1
= 256π 2γ 2 /405 κM 2Sr2 /D 1 + l/r (15)
T2

where D is the diffusivity of water molecules, l is the thickness of an impermeable surface

coating, and κ = V*/C where V* is the volume fraction and C is the total iron concentration.

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Recently, many novel platforms, such as carbon nanotubes (iron oxide-doped),166 zeolites
(Dy(III)-doped),65 and metal-organic frameworks (MOFS) (Dy(III) and Gd(III)-doped)167
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have been studied as potential T2-based contrast agents.

Another type of T2 contrast is derived from chemical exchange and is denoted T2ex.168–169
The effect arises from chemical exchange of protons that have a different frequency than
bulk water. Here the ideal chemical exchange rate (104 – 107 Hz) of protons is slower than
what is required for good T1 relaxation agents.170 Large pseudocontact shift of the protons
that are close to a paramagnetic MRI contrast agent can produce a very large MR frequency
for the proton, that depends on the external magnetic field applied and that can generate a
large T2ex relaxation effect.92 However this mechanism produces relaxivities that are much
lower than dipolar relaxation from Gd(III) or Mn(II) complexes.

3.3.2 Chemical Exchange Saturation Transfer—Comparison of pre- and post-

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injection images allows the detection of enhanced regions using an exogenous relaxation
agent. Ideally these pre- and post-injection images should be acquired during the same
scanning session, otherwise there may be problems of co-registering the pre- and post-
injection data sets because the subject moved. However for targeted imaging and imaging
with nanoparticles it often takes hours for the agent to accumulate at its target and/or for the
background signal to clear. Another potential limitation of relaxation agents is that it can be
difficult to quantify the agent in vivo, especially if the relaxivity changes.171–172
Theoretically, all of these shortcomings can be solved by using chemical exchange
saturation transfer (CEST) agents.45

CEST agents contain a pool of labile protons that can be exchanged with the protons of the
bulk water in an intermediate to slow rate on the NMR time scale (kex ≤ Δω, where Δω is
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the chemical shift frequency difference between the exchangeable pool frequency and the
bulk water frequency.170, 173 A slow water exchange rate is optimal for the production of a
CEST signal at a low radiofrequency pulse for in vivo studies.174 The application of a
presaturation pulse at the frequency of the exchangeable protons leads to the transfer of
some saturated spins into the water pool, which attenuates the signal of the bulk water.
175–177 One of the major advantages of this technique is that despite the small concentration

of the solute molecules (µM to mM range), the signal can be amplified several orders of
magnitude171 through the transmission of the magnetization to the bulk water.178

When a radiofrequency (RF) pulse is applied at the resonance frequency of the labile protons
of the CEST agent then saturation will occur. Because of chemical exchange, the magnetic
saturation will spontaneously be transferred to the bulk water over time (Figure 23A). This
saturation transfer will lead to a decrease in the water signal, through the continuous transfer
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of excited protons to the pool water (Figure 23B, C).179

The CEST effect is sensitive to environmental parameters, such as temperature, pH,

membrane fluidity and cation concentration, as well as the concentration of the CEST agent.
171 The exchange will continue until a steady state is reached or until the RF pulse is turned

off. Using an exchange model consisting of two distinguishable pools (a small solute pool
and large bulk water pool, with no back exchange of saturated protons), the proton transfer

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 Wahsner et al. Page 25

ratio (PTR) in the steady state can be estimated under the assumption that the
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radiofrequency irradiation of the solute pool does not affect the bulk water pool (Equations
16 and 17):176

−tsat
T 1w
PTR = xS ⋅ α ⋅ ksw ⋅ T 1w 1 − e (16)

where

[exchangeable solute protons] kws

xs = = (17)
[water protons] ksw
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According to these equations, the CEST effect increases with the fractional concentration xs,
saturation efficiency α, the exchange rate ksw, and the longitudinal relaxation time of bulk
water T1w.182 The CEST effect depends on the applied field. High fields allow a better
frequency separation in the slow-exchange condition and reduces interference of direct water
saturation. Assuming that transverse relaxation is negligible, the saturation efficiency at a
given power (B1) can be estimated as follows (Equation 18):

2
γB1
α≈ 2
(18)
γB1 2 + ksw
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For analyzing the CEST effect, the most common metric used is the “zero-order-spillover
correction”, called Magnetization Transfer Ratio asymmetry (MTRasym) (Equation 19):

I −ΔCS − I ΔCS
MTRasym ΔCS = (19)
I0

where I(ΔCS) and I(-ΔCS) are signal intensities acquired with RF irradiation applied on-
resonance with the exchanging pool (I(ΔCS)) and at the frequency symmetric around water
(I(-ΔCS)), and I0 is the reference signal intensity acquired without RF pre-saturation. In the
z-spectrum, the normalized water signal intensity is monitored vs the frequency of the off-
resonance saturation.175, 183 The CEST signal is commonly represented as a percent
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decrease in total bulk water intensity. Assuming that the saturation of the bound water signal
is complete, the net magnetization of water protons at steady-state (Mz/M0) can be estimated
as follows (Equation 20):

−1
M z /M 0 % = 100 1 + cqT 1 /55.5τ M (20)

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where c is the concentration of the CEST agent, q is the number of exchanging protons, 55.5
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represents the molar concentration of bulk water, T1 is the relaxation time of bulk water, and
τM is the exchange lifetime of the exchanging proton (1/τM = kex).184

Common CEST agents include amino acids, sugars, nucleotides or heterocyclic rings in their
structure.185 The paramagnetic metal ion present in ParaCEST agents can be a lanthanide
(such as Eu(III), Tm(III), Yb(III)) or a transition metal ion (Fe(II), Co(II) or Ni(II)), with a
short electronic relaxation time. In this case, the election of the macrocyclic backbone as
well as the pendant arms are key to control the oxidation state and the spin state of the metal,
and hence, the stability of the complex.174, 186

There are four categories of exogenous CEST contrast agents: diamagnetic (DiaCEST),
paramagnetic (ParaCEST), liposomal (lipoCEST), and hyperpolarized gas (hyperCEST –
see in section on hyperpolarized molecules).176, 187–188 Even if all rely on exchanging
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protons, the nature of the exchanging protons is different. For example, in the case of
diamagnetic agents (DiaCEST agents), the exchangeable protons usually belong to –NH and
–OH groups, and the chemical shift difference (Δω) with the bulk water protons is less than
6 ppm (Figure 24). This relatively small chemical shift difference requires slow exchange
rates in order to be detected, and this slow exchange usually limits the magnitude of the
CEST effect. In the case of paramagnetic (ParaCEST) agents (chelates that contain
paramagnetic ions), the chemical shift difference (Δω) of the labile protons with the bulk
water protons can be much larger, up to a few hundred ppm, depending on the metal
structure. This larger shift difference means that there can be much faster proton exchange,
but that still obeys the slow exchange condition.171

LipoCEST agents are composed of liposomes that encapsulate a chemical shift agent in the
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inner cavity, thus shifting the resonating frequency of intraliposomal water protons by 1 to 5
ppm from that of extraliposomal water protons (Figure 25).190 The separation in chemical
shift (Δω) between the two proton pools in slow exchange (Δω > kex, kex = exchange rate) is
described in equation 21:

2π ⋅ δBulk water − δIntraliposomal water = Δω (21)

Liposomes present large amounts of equivalent water molecules with exchangeable protons
(106 - 108) in their inner cavities, that are in exchange with the bulk water protons.190 By
introducing a shift agent inside the liposome, the resonance of the intraliposomal water is
shifted relative to the water outside the liposome. The shift agent in LipoCEST agents is
usually a lanthanide complexes in which the ninth coordination site of the metal ion is
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occupied by a water molecule in fast exchange with the intraliposomal water molecules. The
CEST effect is generated by applying a radiofrequency pulse at the resonance frequency of
the intraliposomal water.

HyperCEST is a technique that combines CEST with hyperpolarized MRI, and has been
typically been performed with 129Xe. 129Xe is very soluble in organic solvents and aqueous
solutions (including blood plasma), and possesses a highly polarizable electron cloud (being

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 Wahsner et al. Page 27

highly sensitive to the molecular environment and thus allowing the display of well-resolved
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chemical shifts of solvents, small molecules or proteins it associates with) and can be easily
hyperpolarized, usually using spin-exchange optical pumping (SEOP) where the polarization
is transferred from electronically polarized rubidium atoms in the vapor state to 129Xe
nuclei.191 Because the natural abundance of xenon in the air is at the trace level, no
background signal is observed. 129Xe can be encapsulated in molecular cages that have been
functionalized to bind to a desired target, however direct detection of the hyperpolarized
encapsulated Xe is limited because the Xe can diffuse out of the cage, with only about 1% of
the cage being occupied at a given time.192 Schröder et al. showed that the caged 129Xe
resonance could be saturated and the saturation transferred to the larger unbound 129Xe
resonance in a manner that increased the detection sensitivity over 3300 fold relative to
direct detection of the bound species.193 This had led to various targeted, caged 129Xe
compounds.187, 193–197 A further potential benefit of 129Xe is that it can be inhaled and
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transferred to the blood stream in the lungs. Thus the hyperpolarized signal can be
replenished over time by having the subject breath 129Xe enriched air.

CEST benefits from imaging at high field (7 T and above), since high magnetic fields
increase the chemical shift in Hz, and can improve the specificity of the technique when
several CEST agents are present. Also, at higher fields the T1 relaxation time of water is
increased, making possible a longer accumulation of the saturation of the bulk water, thus
increasing the detectable CEST effect.179

While CEST has several advantages compared to relaxation agents, it also has some inherent
limitations. A primary limitation is the sensitivity which is about two orders of magnitude
lower than relaxation agents.170 In addition, in the body there is an endogenous
magnetization transfer effect that can interfere with the CEST signal. Finally, there are
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limitations to the saturation power that can be applied otherwise dangerous heating of the
subject will occur. Thus while ParaCEST agents with very fast exchanging protons may
exhibit theoretically large CEST effects compared to DiaCEST agents, this gain may not
necessarily be realized in vivo because of the inability to apply enough radiofrequency
power to achieve full saturation.

In order to overcome these shortcomings, Sherry and coworkers prepared europium

complexes with highly shifted exchange sites (large Δω) and very slow water molecule
exchange rates, which allows its activation through relatively low-power RF pulses.
170, 198–201 In general, there is a correlation between the polarity of the ligand pendant arms

and the Eu(III)-bound water lifetime (τM = 1/kex): phosphonates ≥ carboxylates >> alkyl
groups ≥ simple amides.202 For ParaCEST agents, the optimal residence time of the bound
water molecule (τm) is 10−4 – 10−2 s, depending the magnetic field applied during the
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presaturation period. 54

3.4 Thermodynamic stability/kinetic inertness of Gd(III)-based MRI contrast agents

High stability is essential for all metal-based MRI contrast agents used in medicine due to
the well known toxicities of the dissociated metal ions. For example, Gd(III) ions can
irreversibly bind to skeletal tissue and are also known to block Ca(II) binding sites.2, 203

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The prediction of in vivo stability of newly developed Gd(III)-based contrast agents is based
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on fundamental physical properties, like solution thermodynamics, selectivity and

dissociation kinetics. The thermodynamic stability constant KGdL (equation 22) provides the
affinity of Gd(III) for a given ligand and gives the amount of dissociated Gd(III) that is
released in the environment if the system reaches equilibrium.

[GdL]
Gd + L = GdL K GdL = (22)
Gd [L]

The thermodynamic stability constants for the commercially approved GBCAs (Figure 3)
are all large (Table 5), so that the equilibrium lies heavily on the side of the intact complex
GdL. Table 5 shows as well that more basic ligands form thermodynamically more stable
complexes with Gd(III), which is expected given that the Gd(III) ion is a hard acid and
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therefore prefers hard donor atoms. The linear correlation between the sum of the ligand
protonation constants (Σ log K1-n) and thermodynamic stability (log KGdL) was pointed out
many years ago.204 For instance, the DOTA ligand is much more basic than the DTPA-BMA
ligand (sum of ligand protonation constants: Σ log K1-4 = 30.94 vs. Σ log K1-4 = 19.3,
respectively)205 and forms a thermodynamically much more stable complex with Gd(III)
(log KGdL = 24.7 vs. log KGdL = 16.85, respectively). The stability constant does not factor
in competition with other ions or with acid. Very basic ligands will also show strong
competition with proton. The conditional stability constant, Kcond, is a useful number that
gives the equilibrium constant at a given pH, e.g. pH 7.4, and is defined by equation 23,

K GdL
K cond = 2 n
(23)
+
(1 + K 1 H + K 1K 2 H + + K 1K 2K n H +
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where K1, K2, K3, …, Kn are the stepwise protonation constants of the ligand.

Note that [Gd(DOTA)(H2O)]− with the highest basicity among these compounds exhibits the
highest thermodynamic stability constant (24.7211). In contrast, [Gd(DOTA)(H2O)]− exhibits
one of the lowest conditional stability constants (17.2), which stems from its high basicity
and therefore high competition for protons. Note, that the thermodynamic stability constant
and the conditional stability constant for a complex with a less basic ligand like [Gd(DTPA-
BMA)(H2O)] are much closer together (16.9203 vs. 14.8) since there is much less
competition for protons at pH 7.4.206 An alternate value sometimes reported is pM
(−log[M]) which refers to the unchelated metal ion under a given set of conditions ([M]total,
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[L]total, pH), where the higher the pM value, the more stable the complex.

In the body, there is of course competition from other metal ions and from coordinating
anions. There are software programs that can model these systems of equilibria provided the
individual stability constants are all known. In a highly cited paper, Cacheris et al.
introduced a selectivity factor (Ksel), that also considers the stability constants for the
corresponding Zn(II), Cu(II), and Ca(II)-complexes as well as pH (equation 24).79

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K sel

K GdL
= 2 n
(1 + K 1 H + + K 1K 2 H + + ...K 1K 2K n H + + K ZnL[Zn2 +] + K CuL[Cu2 +] + K CaL[Ca2 +]

(24)

The authors found selectivity factors for [Gd(DTPA-BMA)(H2O)] and [Gd(DTPA)(H2O)]2−

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of log Ksel = 9.04 and log Ksel = 7.04, respectively, which reversed the stability trend usually
seen for this two compounds ([Gd(DTPA-BMA)(H2O)]: log KGdL = 16.85 and log Kcond =
14.90 (pH 7.4) vs. ([Gd(DTPA)(H2O)]2−: log KGdL = 22.46 and log Kcond = 17.70 (pH 7.4)).
The authors argued that this higher selectivity value for [Gd(DTPA-BMA)(H2O)] explained
acute toxicity results chelates: LD50 = 14.8 vs 5.6 mmol kg−1 for [Gd(DTPA-BMA)(H2O)]
vs. [Gd(DTPA)(H2O)]2−; LD50 is the dose at which half of the animals die. The authors
claimed that the lower acute toxicity of [Gd(DTPA-BMA)(H2O)] was caused by an
decreased amount of Gd(III) release in comparison to [Gd(DTPA)(H2O)]2−.80 However, in
several studies it was found that actually more Gd(III) is released from administration of
[Gd(DTPA-BMA)(H2O)] than from [Gd(DTPA)(H2O)]2−.213–216 This demonstrates that
care has to be taken when predicting the in vivo stability or toxicity of Gd(III)-based contrast
agents using only thermodynamic stability data.
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In addition to thermodynamics, the rate at which Gd(III) can dissociate from the complex or
be exchanged with an endogenous cation is an equally important factor. In this regard, the
approved macrocyclic contrast agents are much more kinetically inert than the approved
acyclic agents.

Unlike the thermodynamic stability constant, there is no uniform measure of inertness. This
is because different complexes will undergo decomplexation or transmetallation by different
mechanistic pathways. For example, for transmetallation of approved agents with Zn(II), the
rate is first order in [Zn] for acyclic chelates, but is zeroth order for the macrocyclic chelates.
Note, that exchange rate constants are only comparable if the exchange mechanism per
metal complex is the same. Kinetic inertness is reported in various ways. A common
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approach is to measure the dissociation rate constant at a low pH. Another is to measure the
rate of Gd(III) release in the presence of another metal ion or by a competing ligand. Laurent
et al. used a semi-quantitative method where they incubated the chelate with a mixture of
Zn(II) and phosphate.110, 217 Frenzel et al. studied the rate of Gd(III) release from approved
agent when incubated in human plasma.218 Table 6 shows different kinetic measures for
Gd(III) dissociation from commercially approved contrast agents under acidic conditions, in
the presence of Zn(II) and after incubation in human plasma.

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Under all applied conditions, the macrocyclic Gd(III) complexes are the most kinetically
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inert which is in line with what has been observed in in vivo studies where Gd(III) release
was examined in animal models.224 The rigidity of the macrocyclic Gd(III) complexes leads
to considerably slower dissociation rates in comparison to the linear Gd(III) chelates. In
general, it is believed that kinetic inertness is the most critical factor with respect to Gd(III)
release. Among the acyclic chelates, kinetic inertness also appears to predict Gd(III) release
in vivo.213–214, 216, 224–226

It is obvious that novel gadolinium(III)-based MR contrast agents have to meet or exceed the
stability/inertness properties of the approved contrast agents. This is especially important for
targeted and/or macromolecular MRI contrast agents that are expected to have longer in vivo
retention times than simple hydrophilic complexes. Kinetic inertness combined with
thermodynamic stability is the best predictor of in vivo toxicity so far, but the ultimate
standard is how the compounds behave in vivo.
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There is also a strong selection bias in our understanding of contrast agent safety that comes
from the data available for the approved contrast agents. Toxicity may arise from different
causes. While much can be learned from the approved agents, it should be obvious that for
new complexes, increased stability/inertness may not necessarily correlate with safety/
toxicity. Ultimately these compounds would need to be tested in vivo.

4. Contrast agents of improved safety

4.1 Safety concerns
4.1.1 Nephrogenic systemic fibrosis—There is a low incidence of adverse drug
reactions associated with the approved contrast agents and for years these products were
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considered very safe. Prince et al. reviewed data for 160,000 GBCA enhanced MRI studies
and found that acute adverse events occurred at a rate of 5.9 per 10,000 exams, and that
severe adverse events occurred about 1 in 40,000 injections.4 They also mined the FDA
adverse events reporting system database found reports on 40 GBCA associated deaths
between 2004-2009 that were unrelated to nephrogenic systemic fibrosis (NSF) which gave
with an incidence per million doses of 0.15, 0.19, 0.97, 2.7, and 0.7 for gadodiamide,
gadoversetimide, gadopentetate dimeglumine, gadobenate dimeglumine, and gadoteridol,
respectively. In general the use of GBCAs is considered to be very safe.

Throughout the 1990s and early 2000s it was commonplace to administer repeat doses of
GBCA within a short time frame and to administer double and triple dose injections for
angiography or perfusion weighted scans.165, 227 However, in 2006 a strong link between
Gd(III)-based contrast agents and a devastating disease termed nephrogenic systemic fibrosis
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(NSF) was identified in renally impaired patients.5–6 The precise mechanism of NSF onset
remains undetermined but it appears to be strongly connected to exposure to gadolinium.

The risk of NSF increases with diminishing renal function. GBCAs are eliminated almost
exclusively via renal filtration and thus the agent dwells for longer in renally impaired
subject, prolonging the time window for Gd(III) exposure.228–230 Renal function is often
assessed by creatinine glomerular filtration rate (GFR) and the GBCA elimination time

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increases with decreasing GFR. For example, GBCAs typically exhibit blood half-lives on
the order of 90 min in patients with normal renal function (GFR > 90 mL/min/1.73).230–236
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In subjects experiencing moderate renal insufficiency GFR = 30 – 60 mL/min/1.73) the half-

life is increased to 4 – 8 h. In cases of severe renal impairment (GFR < 30 mL/min/1.73 m2)
half-lives ranging from 18 to 34 h have been reported. Gd(III) recovery in the urine of
patients with normal renal fucntion is typically >90% within 12 h of GBCA injection but
recovery decreases with decreasing renal function. For example, Gd(III) recovery as low as
76% after 5 days was reported for patients with GFR under 10 mL/min/1.73 m2230. NSF
incidence among the 8 FDA approved contrast agents appears to reflect the relative kinetic
inertness of the Gd(III) complex as determined by in vitro assays.237–238

Following the linkage between NSF and gadolinium exposure, regulatory agencies like FDA
placed restrictions on the use of GBCAs. In 2010 FDA contraindicated the use of Gd-DTPA,
Gd-DTPA-BMA, and Gd-DTPA-BMEA in patients with GFR < 30 mL/min/1.73 m2, and
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recommended to test for GFR and to avoid use of all GBCAs in patients with impaired renal
function.239 The American College of Radiology (ACR) 2015 manual recommends GFR
screening of any patients with known or suspected renal impairment.240 The ACR also
recommends extreme caution in administering any Gd(III) contrast agent to patients of GFR
< 40 mL/min/1.73 m2, suffering acute injury, or in need of dialysis. These guidelines are
necessary and have essentially eliminated new incidences of NSF.

4.1.2 Brain and body retention of Gd(III)—Mounting evidence since 2013 indicates
that Gd(III) from contrast agents is irreversibly retained in the central nervous system
(CNS). Concerns were first raised after reports of prolonged and non-diminishing signal
enhancement in the dentate nucleus and globus pallidus of patients receiving Gd(III) contrast
agents.241 The relative degree of CNS enhancement correlated to the number of contrast
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enhanced examinations received.242 A 2015 analysis of autopsy specimens confirmed that

the CNS enhancement was indeed due to Gd(III) deposition.7 As with NSF risk, the risks of
CNS Gd(III) accumulation associated with each agent appear to reflect kinetic inertness – a
greater degree of CNS accumulation is observed following exposure to the less kinetically
inert linear agents.243–244 However, significantly elevated signal in the dentate nucleus has
also been observed in Multiple Sclerosis patients exclusively receiving multiple doses of the
macrocyclic agents Gd-DO3A-butrol or Gd-DOTA.245–246 Here too, dentate nucleus signal
enhancement reflects cumulative dose. A conflicting study did not detect any significantly
elevated signal in the dentate nucleus or globus pallidus of a brain tumor patients receiving
multiple doses of Gd-DO3A-butrol exclusively.247 The long term effects of Gd(III)
deposition in the CNS remain unknown and no toxic effect has yet been identfied. However,
these new findings have triggered a high degree of anxiety about the safety of Gd(III)-based
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agents and stimulated a new wave of regulatory activity and debate.

Gadolinium deposition has also been identified in the body of patients with normal renal
function after receiving GBCAs. Gd(III) retention has been identified in femoral heads
extracted from patients receiving hip replacement surgery who had previously received Gd-
DTPA-BMA or Gd-HPDO3A.248 Bone retention was 2.5 greater in the patients who
received Gd-DTPA-BMA. A 2016 autopsy study demonstrating elevated bone levels of
gadolinium by mass spectrometry in adult patients after receiving linear or macrocyclic

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GBCAs.249 A 2018 study quantified gadolinium retention in tibeas of healthy normal

volunteers 5 years after a single dose of Gd-DO3A-butrol using X-ray fluorescence.250 The
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study identified long term bone accumulation that increased with cumulative dose. An
average accumulation of 2.5 nmol Gd(III) per g bone mineral for every mmol/kg GBCA
administered was observed. Another study identified a positive correlation between liver
gadolinium and iron concentrations in pediatric patients with iron overload after receiving
Gd-DOTA.251

A few studies have attempted to discern the speciation of accumulated Gd(III). Phosphate
bound Gd(III) was identified by extended X-ray absorbance fine structure (EXAFS) analysis
in the skin of NSF patients, which is consistent with dechelated Gd(III).252 In another study,
water soluble Gd(III) was extracted from the biopsied skin of NSF patients and analyzed by
HPLC-ICP-MS.253 The water soluble Gd(III)-containing species was identified as Gd-
HPDO3A. After extraction of the water soluble Gd(III)-containing species the skin sample
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was analyzed by laser ablation ICP-MS which showed that the distribution of insoluble
Gd(III) was strongly correlated with phosphorous distribution.253 A study of the speciation
of accumulated gadolinium in the brains of rats after repeat exposure to high doses of
commercially available GBCAs revealed that Gd(III) accumulated after treatment with linear
agents existed in 3 forms: intact chelate, soluble macromolecule associated Gd(III), and
insoluble Gd(III) deposits, whereas Gd(III) accumulated after treatment with macrocylic
agents existed solely as intact chelate.254 Another study comparing speciation of Gd(III)
retained in the rat brain after repeat administration of Gd-DOTA and Gd-DTPA-BMA
demonstrated that Gd(III) from Gd-DOTA exists as the intact chelator where Gd(III) from
Gd-DTPA-BMA is predominatly recovered as insoluble Gd(III).255

Chelation therapy has been considered as a method to remediate Gd(III) accumulation.

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Levels of Gd(III) accumulated in pediatric patients suffering iron overload after Gd-DOTA
treatment was shown to decrease after iron chelation therapy.251 An oral chelator
formulation is currently under development as a Gd(III) remediation treatment.256

There have also been reports of NSF-like and neurologic symptoms in patients with normal
renal function who have received GBCAs.257–259 However these cases have primarily been
identified through patient self-reporting and the linkage to gadolinium remains controversial.

4.1.3 Current regulatory status—In 2010, the FDA forbade the use of the 3 least
kinetically inert agents (Gd-DTPA, Gd-DTPA-BMA, Gd-DTPA-BMEA) in patients with
GFR ≤30 mL/min/1.73 m2. 238–239 The FDA also labelled all contrast agents with a boxed
warning that advises against exceeding the recommended dose and against repeat dosing.
238–239 Following the reports of brain deposition, the European Medicines Agency
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announced in 2017 that it would suspend the marketing authorization for 3 of the 8 available
intravenous formulations (Gd-DTPA, Gd-DTPA-BMA, Gd-DTPA-BMEA), while limiting
the use of Gd-DTPA-EOB and Gd-BOPTA to liver imaging applications.260 FDA did not
recommend any product withdrawals but did add new warning labels advising caution and
prudence when ordering an MRI scan.261–262 Japan’s Pharmaceuticals and Medical Devices
similarly updated their GBCA package inserts.

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Limiting the use of GBCAs in patients with impaired renal function eliminates NSF risk but
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creates challenges in patient management. For instance an estimated 16% of US adults suffer
chronic kidney disease stage 3 (GFR ≤60 mL/min/1.73 m2) or greater.263 Besides their
kidney disease, these patients often have other co-morbidities like diabetes or cardiovascular
disease.264–265 Physicians managing these patients must now carefully weigh the risk versus
benefit of contrast enhanced examinations that would otherwise be routine for patients with
normal renal function. There are often no good clinical alternatives available to replace
GBCA enhanced scans. Intravenous X-ray contrast agents, which can be used for blood
vessel imaging also poses a potentially severe nephrotoxicity risk to renally impaired
patients.266

The current concerns over the safety of MRI contrast agents is driving chemistry innovations
towards the design of imaging products of enhanced safety. Four main approaches have been
taken. One approach is to focus on developing higher relaxivity gadolinium(III) based
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contrast agents which can be administered at a lower dose than current GBCAs. A second
approach is to identify Gd(III) complexes that are even more inert to Gd(III) dissociation/
transmetallation. A third approach is to use molecular targeting to improve specificity and
lower the dose. Another approach is to develop a next generation of MRI contrast agents that
moves away from Gd(III) altogether. We will review progress towards both these goals
below.

It should be kept in mind though that GBCAs have a very good safety profile over all, and so
far it is better than the more limited experience with iron oxide based nanoparticles. For
instance with the iron oxide ferumoxytol, the FDA found 79 anaphylactic reactions in its
Adverse Event Reporting System between June 2009 and June 30, 2014, involving patients
aged 19 to 96 years.267 Half of the reactions occurred with the first dose of ferumoxytol, and
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three-quarters began during infusion or within 5 minutes of completion. Eighteen of those 79

patients died despite immediate medical intervention. The incidence rates of anaphylaxis or
serious hypersensitivity reaction varied between 0.2% and 0.9%, depending on the patient
population in clinical trials of ferumoxytol.268 This is in the range of 2 – 10 serious events
per 1000 injections. On the other hand GBCAs have a more favorable safety profile, with a
rate of 0.04 serious events per 1000 injections (40 per million). Over a 5 year reporting
period the total number of deaths associated with GBCAs was 41 deaths out of 51 million
GBCA administrations (0.9 deaths per million administrations). New compounds will have
to meet a high safety standard.

4.2 High relaxivity Gd(III)-based contrast agents

There are currently 7 Gd(III)-based ECF agents that are FDA approved and these have r1
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values in blood plasma in the range of 3.6 to 6.3 mM−1s−1 at 1.5 and 3T. For clinical use, the
approved dose range from 0.1 to 0.3 mmol kg−1. For those agents, it is estimated that the
local concentrations of Gd(III) must be around 125 µM to robustly observe contrast
differences in tissue (this corresponds to a relaxation rate change of about 0.5 s−1).
Regarding absolute sensitivity, the detection limit for the MRI contrast agent Gd-HPDO3A
(Figure 3) in mouse skeletal muscle was reported to be 30 µM.269 Hence, for targeted MR

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contrast agents with similar T1 relaxivity values as the commercial contrast agents, the
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biological target should be present in excess of 100 µM.

Research has focused on improving sensitivity and expanding the scope of clinical MR
contrast agents for higher efficiency, without sacrificing safety. Relaxivity is a measure of
the sensitivity of the contrast agent. The typical T1 relaxivity values of commercially
available gadolinium(III) based contrast agents are relatively small compared to what is
theoretically possible.270 Contrast agents with higher relaxivity could provide greater tissue
enhancement for better detection of smaller lesions, or could produce equivalent contrast at a
lower dose compared to existing approved compounds, which may lower the risk of Gd(III)-
induced toxicity. For targeted or activatable contrast agents, using high relaxivity compounds
would also allow the detection of lower concentration targets.

Several molecular parameters that govern the T1 relaxivity of MR contrast agents need to be
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tuned to achieve higher relaxivity.271 Such parameters are the hydration number (q), the
rotational correlation time (τR) and the water residency time (τm). An optimum relaxivity
can be reached if the hydration number q is increased (q = 1 in approved contrast agents), if
the rotational correlation time (τR) relative to commercial CAs is increased,270 and if the
coordinated water residency time τm is decreased to 1-30 ns (τm = 150-1000 ns for approved
contrast agents).

4.2.1 Increasing the hydration number q of Gd(III)-based contrast agents—

Inner-sphere relaxivity is directly proportional to hydration number. There has been
considerable effort to generate coordination cages capable of forming highly stable and
kinetically inert Gd(III) complexes (inert with respect to Gd(III) dissociation) with an
extended inner-sphere hydration number. The hydration number can be increased by
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lowering the denticity of the multidentate co-ligand, e.g. using heptadentate instead of
octadentate chelators. However, this often reduces the thermodynamic stability and/or
kinetic inertness of the resulting Gd(III) complex, and increases the risk of Gd(III) release
into the body. Increasing the hydration sphere also raises the risk of anion binding and
displacement of coordinated water ligands. For instance, the stable q = 2 complex Gd-DO3A
can form ternary complexes with endogenous coordinating anions such as phosphate or
bicarbonate.272 Anion coordination results in the displacement of the coordinating water
ligands, and as a result the T1 relaxivity is reduced.

Figure 26 shows 5 ligand systems that form stable gadolinium(III) complexes with increased
hydration number: the heptadentate chelators PCTA, AAZTA, CyPic3A, and aDO3A and the
hexadentate chelator tacn(1-Me-3,2-hopo)3. Gd-PCTA is thermodynamically less stable than
Gd-DOTA (log Kcond = 16.2 vs. log Kcond = 17.2),206 but it is kinetically quite inert. In acid-
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assisted gadolinium decomplexation experiments Gd-PCTA reacts only an order of

magnitude faster than Gd-DOTA, which makes it superior to the approved macrocyclic
contrast agents Gd-HPDO3A and all commercially approved acyclic contrast agents. Due to
its two metal-bound water molecules, the T1 relaxivity of Gd-PCTA (r1 = 6.9 mM−1s−1 at 20
MHz, 25 °C) is higher than that of the approved ECF agents.273–276

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Gd-AAZTA (log Kcond = 16.4)206 is also thermodynamically less stable than Gd-DOTA, but
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its kinetic inertness may be sufficient for in vivo applications. Although it was shown that
Gd-AAZTA reacts faster in acid-catalyzed decomplexation experiments than Gd-DTPA, it
undergoes transmetallation reactions with Cu(II) or Eu(III) more slowly than Gd-DTPA.277
Its hydration sphere features two water molecules leading to a high T1 relaxivity of 7.1 mM
−1s−1 (20 MHz, 25 °C).278–280

Gd-CyPic3A affords two metal-coordinated water molecules (q = 2) and an r1 value of 5.70

mM−1s−1 (60 MHz, 37 °C), which is 1.7 fold higher than that of Gd-DTPA measured under
the same conditions (r1 = 3.26 mM−1s−1). Its thermodynamic stability is comparable to that
of the approved contrast agent Gd-HPDO3A and its kinetic inertness is in the range of
acyclic clinically used Gd(III) complexes. It was also shown that the complex is resistant to
anion coordination.281
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Parker and coworkers synthesized Gd-aDO3A, a bishydrated Gd-DO3A derivative with

remarkably high relaxivity values (r1 = 12.3 mM−1s−1 at 20 MHz, 25 °C). Unlike Gd-DO3A,
Gd-aDO3A is not prone to inner-sphere water displacement by endogenous anions,
presumably because of repulsion by the negatively charged pendant carboxylate groups. The
kinetic inertness of this complex was reported to be 10 times higher than that of Gd-DTPA.
282

Raymond and coworkers developed a series of thermodynamic stable Gd(III) complexes that
utilized three hydroxypyridinone (HOPO) bidentate chelators. These complexes exhibited
hydration numbers of up to q = 3 and hence, they were able to obtain exceptionally high T1
relaxivity values (e.g. 13.1 mM−1s−1 at 20 MHz, 25 °C for Gd-tacn(1-Me-3,2-hopo)3).
Moreover, those complexes are resistant to anion coordination.283–287
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Recently, Fries and coworkers reported a comparative study of the experimental high
relaxivity agent P03277 (Figure 27, left) with the approved contrast agent gadobutrol (Figure
3).288 P03277 is a Gd-PCTA derivative with a low molecular weight (0.97 kDa) and a
measured value of q = 1.7. Its T1 relaxivity in human blood plasma at 37 °C is
approximately 2.5 times higher than that of gadobutrol at clinical field strength (12.8 vs. 5.2
mM−1s−1 (1.5 T) and 11.6 vs. 5.0 mM−1s−1 (3.0 T), respectively). Increases in relaxivity due
to increased hydration should be maintained at high fields, and P03277 has 2-fold higher
relaxivity than gadobutrol (9.3 vs. 4.8 mM−1s−1 at 7.0 T, 8.6 vs. 4.7 mM−1s−1 at 9.4 T,
respectively in human plasma, 37°C). An in vivo comparison of both agents in a rat model of
hepatic colorectal cancer metastases at 9.4 T revealed that P03277 has similar
pharmacokinetics as gadobutrol but demonstrates significantly better tumor enhancement
properties as depicted in Figure 27, right. P03277 is currently undergoing human clinical
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trials (NCT02633501, www.clinicaltrials.gov).

4.2.2 Optimizing the rotational correlation time (τR) of Gd(III)-based contrast

agents—As discussed above, r1 for simple chelates is limited by fast rotation at all
accessible field strengths above 0.1 T. Slowing down the rotational motion of the complex is
an effective way to increase relaxivity. This can be achieved by increasing the molecular
weight of the contrast agent. This in turn can be achieved by making the complex larger, or

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by the covalent or non-covalent binding of the contrast agent to a macromolecule (e.g. a

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protein, a polypeptide, a nanosphere, or micellar nanoparticles). The linear dependence of

the molecular weight of monohydrated Gd-DOTA derivatives and their T1 relaxivities (0.5 T,
25 °C) is illustrated in Figure 28, left.289 Similarly, the rotational correlation time exhibits
the same linear correlation with the molecular weight of those Gd(III) complexes Figure 28,
right.

In order to attach paramagnetic agents to macromolecules, bifunctional chelates have been

developed.290 Typically, they are based on the chelators DOTA or DTPA and are bearing an
electrophilic group, such as isothiocyanates, N-hydroxysuccinimide ester, maleimides etc.,
that is available for conjugation to nucleophilic groups on macromolecules. Excellent
reviews are available, in which those agents are covered.291–293

In an early example, Gd-DTPA chelates were covalently attached to HSA yielding the
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macromolecular contrast agent albumin-(Gd-DTPA)19 with a molecular weight of 92 kDa.

The T1 relaxivity per Gd(III) ion was found to be 3 times higher than that of the monomeric
chelate (20 MHz, 37 °C), which is attributed to the larger molecular weight of the contrast
agent and hence, its higher rotational correlation time.294–296 Gd-DTPA has been also
conjugated to poly-L-lysine.297 The resulting macromolecular compound (average
molecular weight: 49 kDa) possesses a r1 value that is also 3 times higher than that of the
monomeric chelate.298

Aside from tethering a Gd(III) complex to a macromolecular structure through a linker,

peptides or proteins were designed with embedded Gd(III) binding sides. Franklin and
coworkers prepared the peptide chimera GdP3W, a 4 kDa metallopeptide with an EF-hand
Gd(III) binding site and DNA binding domain.299 The rigid structure and extended
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hydration sphere (q = 2) of GdP3W furnishes a r1 value that is 6 times higher than that of
Gd-DTPA (60 MHz, 37 °C). Moreover, a 100 % increase in its T1 relaxivity was observed
upon binding to DNA.

Another example are the protein-based Gd(III) MRI contrast agents (ProCAs).300 Yang et al.
showed that engineered proteins chelated with Gd(III) (average molecular weight: 12 kDa)
have relaxivities that are 20-times higher than that of Gd-DTPA, and are inert to Gd(III)
release.301 Protein engineering with tumor targeting modalities allowed imaging of e.g.
prostate or liver cancer in mouse models.302–303

Another strategy to increase the molecular weight of the MR contrast agent is to link
multiple Gd(III) complexes together, which in turn also enhances the number of Gd(III) ions
per molecule and therefore the overall contrast. In this approach, the coupling of multiple
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Gd(III) compounds to spherically shaped carriers, e.g. dendrimers, is more effective than
linking Gd(III) complexes in a linear fashion. Although there is internal motion in both
instances (see below), dendrimers rotate in a more isotropic fashion whereas linear
oligomers rotate anisotropically. Linear oligomers will rotate more rapidly about their long
axis and this faster rotation will limit relaxivity. A Gd-DO3A derivative assembled to a
spherical dendrimer59 has 40% higher relaxivity per gadolinium ion than the same Gd-
DO3A derivative assembled to a linear dextran polymer,304 although the molecular weight

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of the linear polymer (52 kDa) is higher than that of the dendrimer (17 kDa). The different
types of motion are depicted in Figure 30.109
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Internal Motion: The nuclear relaxation induced by the chelate is dependent on the rotation
about the M-H(water) vector. To this point we have considered that the rotational motion of
this vector is isotropic and mirrors that of the complex. This is a good approximation in
many cases. But there are often internal rotations that are occurring faster than the overall
reorientation of the entire molecule. The simplest of these is rotation about the M-O bond.
Over 40 years ago, Dwek pointed out that for water coordinated to a metal ion, fast rotation
about the metal-oxygen bond will decrease the efficiency of the paramagnetic relaxation
effect.305 For MRI contrast agents, this fast intramolecular motion will reduce the relaxivity
of the complex.

Direct experimental assessment of water rotation is challenging. Using the Ln-DOTAM

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system where the water exchange rate is very slow, Dunand et al. were able to measure the
17O and 1H relaxation times of the coordinated water for the Eu(III) complex and determine

the effect of this fast rotation.306–307 However water exchange in most lanthanide complexes
is too fast for direct observation of the coordinated water ligand. Nonetheless, this fast
intramolecular rotation is assumed to occur in many Gd(III) complexes, including those used
as commercial MRI contrast agents.144, 293 The effect of this fast rotation is to reduce inner-
sphere relaxivity by about 30%.

Recently Boros et al. showed that this fast water rotation could be reduced by the formation
of an intramolecular hydrogen bond between the coordinated water hydrogen and a suitable
H-bond acceptor (Figure 31), and that the resultant complex would have a higher relaxivity
than similar compounds lacking the H-bond acceptor.123 In that study the authors used Gd-
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DOTAla derivatives with pendant H-bond acceptors as well as control compounds that were
structurally very similar but lacking this H-bond acceptor. There are several reports of
gadolinium(III) complexes having anomalously high relaxivity compared to related
complexes of similar size, shape, and charge. For instance, work of Lowe et al. describes
Gd(III) complexes with a pH-dependent, intramolecular ligation of a β-arylsulfonamide
nitrogen which results in changes in the hydration state at the lanthanide center.308 The
relaxivity observed for the q = 2 complexes was found to be anomalously high, and possibly
indicative of interaction with the bound water by pendant, non-coordinating carboxylates.
Similarly, Dumas et al. described Gd(III) complexes with unexpectedly enhanced relaxivity
that possess pendant non-coordinating carboxylate donor arms.309–310 Previously, these
cases of high relaxivity have usually been ascribed to a second-sphere effect wherein water
molecules in the second hydration sphere have extended residency times because of
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interactions with H-bond acceptor(s) on the chelating ligand. However in many of those
complexes it is likely that the H-bond acceptor could also be interacting with the inner-
sphere coordinated water and hindering the water rotation.

For more complicated molecules such as multimeric agents or chelates (non)covalently

attached to a macromolecule, there can be additional internal motions that reduce relaxivity.
For example if the chelate is covalently attached to a macromolecule by a linker group, then
the chelate can rotate about this linker so that the rotation of the chelate is decoupled from

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the macromolecule and the relaxivity is not as high as predicted based on the overall motion
of the macromolecule.311–314 For these complex systems where there are a number of
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internal motions, the data are usually analyzed by a model free approach described
originally by Lipari and Szabo.315 They showed that the usual spectral density function for
isotropic motion could be replaced by two terms, one that reflected the overall rotation (or
global motion (τg)) of the molecule and another that reflected internal (or local) motions (τl)
(equation 25),

1 C 3Fτcg 3(1 − F)τcl

= 6 ⋅ + (25)
T 1m r 1 + ω2H τ2cg 1 + ω2H τ2cl
GdH

1 1 1 1
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= + + (26)
τcg τg T 1e τm

1 1 1
= + (27)
τcl τcg τl

with C as a constant (as in equation 9) and F as a measure for the degree of isotropic motion.

The relative contributions to the global and local motions is given by an order parameter S2
(sometimes denoted F to avoid confusion with the spin quantum number). At one extreme,
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the rotational motion of the chelate is completely decoupled from the macromolecule (F =
0). On the other extreme (F = 1), the chelate is immobilized on the macromolecule such that
internal motion is prevented, and the spectral density function is the same as for isotropic
rotation. In Figure 32 the effect of internal motion on relaxivity is simulated for a system
with a very long rotational correlation time (10 ns) associated with a macromolecule and a
local rotational correlation time of 0.1 ns at proton Larmor frequencies ranging from 1-100
MHz. Figure 32 shows that internal motion can dramatically reduce relaxivity. However the
effect of internal motion on relaxivity is more pronounced at lower fields (<1 T).109

Strategies to minimize internal motion: One effective strategy to minimize internal motion
is to force the metal-water proton vector to rotate isotropically with the entire molecule.
Parker and coworkers showed that this can be done by placing metal at the molecular
barycenter (Figure 30) to create contrast agents with low molecular weight (3 – 7 kDa) but
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still remarkably high relaxivity in the region of 20 – 40 mM−1s−1.31, 316–317 In these studies
they used 4 large hydrophilic pendant arms to increase the molecular size of Gd-DOTA
derivatives. The blood pool agent P792 utilized the same strategy.32

Another approach is to limit internal motion by connecting the chelate to the

macromolecular structure by two points of attachment. Early examples were the self-

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assembled metallostar compounds, where the rigid and multimeric structure of the MR
contrast agent is formed in the presence of Fe(II) ions.318–320
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In another example, Zhang et al. combined a multimeric approach with protein binding to
boost relaxivity.321 They synthesized a tetrameric Gd-DTPA oligomer with two HSA
binding groups (Figure 33). In the absence of protein, the relaxivity is relatively low because
of the flexible multimeric structure. But upon binding to HSA the contrast agent is rigidified
and rotational motion slowed, dramatically increasing relaxivity. This is shown
schematically in Figure 33. For small molecules like MS-325, relaxivity is low (Figure 33,
A) but is increased several fold upon protein binding (Figure 33, B). However for multimeric
agents, the gain in relaxivity upon protein binding is often offset by the internal motion
(Figure 33, C). But using a dual anchor strategy overcomes this relaxivity loss (Figure 33,
D)). Indeed, the authors obtained an enhanced r1 of 39.1 mM−1s−1 (20 MHz, 37 °C, pH 7.4)
for their dual binding Gd(III) tetramer in the presence of HSA (Figure 33, E)). This was
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50 % higher than the relaxivity obtained for the same tetramer with only one HSA binding
unit (schematic illustration in Figure 33, C)) where r1 = 26.2 mM−1s−1 measured under the
same conditions. On a molecular basis, the relaxivity is 4 times higher.

Another example of a dual anchor strategy applied to liposome based contrast agents was
reported by Botta, Tei, and coworkers.322–323 Amphiphilic Gd(III)-chelates can be
incorporated into liposomal bilayers resulting in increased relaxivity. However if the chelate
has one alkyl chain for incorporation, significant internal motion can occur. The authors
used Gd-DOTA(GA)2 derivatives that had near optimal water exchange kinetics and two
points of attachment to the lipid bilayer. As a result relaxivity was doubled compared to the
analogous compound with only a single point of attachment.
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Contrast agents with very long rotational correlation times (on the order of 10 ns) can have
near optimal relaxivity at low fields (0.5 – 1 T), but for these types of compounds the
relaxivity falls off dramatically at higher field strength. For higher field strengths, one
requires an intermediate rotational correlation time (on the order of 1 ns) which will result in
very good relaxivity at 1.5 T and close to optimal relaxivity at 3 T and 7 T.

As an example, Figure 34 shows the influence of the magnetic field strength on the T1
relaxivity of three compounds with different rotational correlation times: Gd-HPDO3A
(short τR, see Figure 3), Gd3L3 (intermediate τR, see Figure 35), and MS-325 bound to HSA
(long τR, see Figure 3).324 Gd3L3 employs the Gd-DOTAla chelate which allows it to be
incorporated into a peptide structure via two points of attachment, thereby limiting internal
motion. Figure 34, A presents r1 per molecule and Figure 34, B presents r1 per Gd(III). At
0.47 T, the compounds with intermediate or long τR exhibit similar molecular T1
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relaxivities, but with increasing field strength (here, to 11.7 T), the difference between those
agents becomes larger. Gd3L3 with its intermediate correlation time is superior to HSA-
bound MS-325, 50 % higher at 1.5 T and 350 – 450 % higher at 4.7 – 11.7 T. On a per
Gd(III) basis, Gd3L3 has 50 – 220 % higher r1 than HSA-bound MS-325 at high fields. Note
that the T1 relaxivity of Gd-HPDO3A (short τR) stays roughly the same at all field strengths.

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Since clinical imaging is increasingly moving to higher field strengths, it is important to

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develop contrast agents that are applicable at high field. For 3 – 7 T, a rotational correlation
time in the range of 0.5 – 2 ns is desirable. Still, it should rotate isotropically with limited
internal motion. Recently, several examples for high field Gd(III)-based MR contrast agents
were reported.320, 324–330

It should be noted that the same strategies apply to Mn(II), Fe(III), and Mn(III) chelates.
Slowing down rotation by protein binding results in large increases in relaxivity at lower
field strengths.331–333 Similarly, rigid multimers with multiple chelates can provide
intermediate rotational correlation times that provide enhanced high field relaxivity.334

4.2.3 Tuning the water residency time (τm) of Gd(III)-based CAs—Another

parameter that can be tuned to increase r1 is the water residency time (τm). The coordinated
water must be in rapid exchange with bulk solvent in order to transmit the relaxation effect
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to the solvent. Relaxivity can be limited if τm is too slow or too fast. It is important to note
that for fast tumbling complexes T1m >> τm, and so changing τm has little to no effect on
relaxivity. However once the rotational correlation time begins to be optimized, then τm can
play a major role.

As an example, optimal values for contrast agents with a water residency time similar to Gd-
DTPA or Gd-DOTA (τm ~100 ns at 37 °C) are around 10 ns (Figure 36, left). However,
incorporation of the gadolinium(III) compound into a macromolecule might affect the other
parameters that influence the relaxivity. MS-325 is a Gd-DTPA derivative that can non-
covalently bind to the plasma protein human serum albumin (HSA) (Figure 36, right).
MS-325 (unbound) has a τR of 115 ps. Once bound to HSA, MS-325 (bound) has a τR of
10.1 ns, which causes a dramatic increase in T1 relaxivity. However, the observed T1
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relaxivity is not as high as it theoretically should be, because another parameter that governs
relaxivity, the water residency time (τR) is influenced by this modification. Indeed, MS-325
(unbound) has a τR of 69 (± 20) ns and MS-325 (bound) has a τR of 170 (± 40) ns. The
water residency time becomes crucial when the rotational motion is slowed, which limits the
observed T1 relaxivity.17 This shows that the lability of the water molecule at the metal side
can be influenced when a supramolecular adduct is formed.335

The effect of water exchange of slow tumbling systems is also seen when comparing
MS-325 and its bis(N-methyl)amide derivative: MS-325-BMA (Figure 37). Gd(III)
complexes with amide oxygen donor atoms are known to have slower water exchange rates
compared to the analogous carboxylate derivatives.111 The water exchange rate of MS-325-
BMA is 10 times slower than the water exchange rate of MS-325. Since both MS-325 and
MS-325-BMA have a similar size, their rotational correlation times and T1m values should
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be similar in phosphate buffered saline (PBS) or when bound to HSA. In PBS the relaxivity
of these complexes is alike indicating that T1m >> τm. However, once bound to HSA the
relaxivity of MS-325 is 3 times higher than that of MS-325-BMA. When bound to HSA,
T1m is shortened to the point that the long τm of MS-325-BMA limits its relaxivity.109

The water exchange rate can be accelerated by increasing the steric compression around the
water binding site. This was originally demonstrated by Merbach and co-workers in

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octadentate chelates, such as Gd-DTPA (Figure 3) and Gd-EPTPA (Figure 39).

111, 143, 336–338
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However, for these Gd-DTPA derivatives, the rate enhancement is achieved

at the cost of lower thermodynamic stability.

The steric crowding in Gd-DOTA derivatives can also be enhanced by introducing bulky
groups. Dumas et al. found that exchanging one acetate arm by a different donor group led
to an increase in τm in the following order: phosphonates ~ phenolates < alpha-substituted
acetates < acetate < hydroxamates ~ sulphonamides < acetamide ~ pyridyl ~ imidazole,
which is also in line with the general trend that neutral donors slow down water exchange
kinetics in comparison to negatively charged donor groups.112 In these complexes, the
coordinated water is released through a dissociative exchange process that is favored by
steric crowding.270

In total, Dumas et al. reported 38 Gd-DOTA derivatives with HSA binding groups, and the
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water residency time varied over 3 orders of magnitude. In the absence of HSA, r1 was
similar for all compounds with a given q values and correlated with molecular weight. As
expected the relaxivity did not correlate with τm. The influence of τm on relaxivity becomes
apparent when the relaxivity was measured in the presence of HSA. Slow tumbling shortens
T1m and now τm can limit relaxivity. Figure 38 shows the relaxivity for this series of
chelates bound to HSA as a function of their water residency time at two different field
strengths (0.47 T and 1.4 T). As predicted by theory there is an optimal water residency time
at low field for slow tumbling compounds, which peaks between 10 ns and 30 ns (0.47 T).
When increasing the field strength to 1.4 T, the range of τm for optimal relaxivity becomes
broader.

With phosphonate groups in particular, the steric crowding combined with the favorable
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arrangement of the water molecules in the second coordination sphere leads to a faster water
exchange rate.340 For instance, Gd-DO3A-pyNox (Figure 39), a Gd-DO3A derivative
bearing a sterically demanding group has a water residency time of 39.0 ns (25 °C) while
Gd-DOTA has a water residency time of 244 ns (25 °C).341 The water residency time in a
monophosphinic acid derivative Gd-DO3APABn (Figure 39) was found to be even faster
with τm = 16.2 ns (25 °C).340 Boros et al. recently showed that for slow tumbling systems
with exceptionally fast water exchange that the water residency time becomes the correlation
time influencing relaxvity and results in a useful strategy to achieve high field, high
relaxivity.342

4.2.4 High relaxivity Gd(III)-nanomaterials—Gd(III)-nanomaterials have been the

subject of intensive research for the past two decades since they have unique potential to
overcome the sensitivity disadvantage of contrast agents. Their ability to carry high Gd(III)
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payloads combined with their long rotational correlation times makes them excellent
candidates in the design of high relaxivity MRI contrast agents. Additionally, these materials
offer great flexibility in terms of surface modifications for e.g. multifunction or
bioconjugation for therapeutic or diagnostic applications, respectively.

Nanoparticles engineered as MRI contrast agents have been based on carbon nanotubes and
fullerenes, polymers and dendrimers, gold, silicon, viral nanoparticles, and liposomes and

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micelles. A comprehensive overview of the recent progress on Gd(III)-nanomaterials as MRI

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contrast agents is beyond the scope of this review. Excellent reviews are available that cover
those agents.343–347

An impressive example for a Gd(III)-nanosystem with exceptional high relaxivity was

reported by Wilson and coworkers. They developed gadonanotubes (Gd3+n@US tubes), that
consist of aquated Gd(III)-ion clusters confined within single-walled carbon nanotube
capsules.348 At clinical field strength, the r1 values were found to be 40 times higher than
that of the Gd(III)-based ECF contrast agents (r1 = ~170 mM−1s−1 per Gd(III)-ion, 60 MHz,
40 °C).

Meade and coworkers took a different approach and conjugated Gd-DO3A derivatives to the
surface of nanodiamonds.349 The resulting Gd(III)-nanodiamond conjugate affords a T1
relaxivity that is 10 times higher than that of the monomeric Gd(III)-complex (r1 = 58.8 mM
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−1s−1 per Gd(III) ion, 60 MHz, 37 °C).

In a recent example, it was shown that water permeable silica nanoparticles with confined
monohydrated Gd-ebpatcn chelates (Figure 39) feature a nanosized contrast agent with an
extraordinary high r1 value of 84 mM−1s−1 at 35 MHz (25 °C).350

In another example, Chuburu and coworkers incorporated Gd-DOTA chelates into a

polysaccharide-based hydrogel (Gd-DOTA-NPs).351 As illustrated in Figure 40, the T1
relaxivity of the Gd(III)-loaded nanoparticle was found to be 24 times higher than that of the
monomeric Gd-DOTA complex (r1 = 72.3 mM−1s−1 per Gd(III)-ion, 60 MHz, 37 °C).

4.3 Increasing resistance to Gd(III) release

Trivalent gadolinium is an oxyphilic hard cation that favors coordination numbers of 8 and 9
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with negatively charged oxygen atom donors preferred to neutral oxygen donor atoms. This
becomes apparent by comparing the stability constants of [Gd(DOTA)(H2O)]− and [Gd(HP-
DO3A)(H2O)], in which a negatively charged acetato donor was replaced by an uncharged
hydroxyl donor (Figure 3; log KGdL = 24.7 vs. log KGdL = 23.8, respectively).

The formation of 5-membered chelate rings is generally preferred, whereas the chelate ring
size is one of the dominating factors that govern stability. It is well known that changes in
chelate ring size greatly influence stability. As an example introducing one or two additional
methylene units in the macrocyclic backbone of Gd-DOTA gives Gd-TRITA and Gd-TETA
(Figure 41), respectively and causes a drop in thermodynamic stability in 5 orders of
magnitude each.206 Such a drop in stability can also be observed by changing the pendant
acetato moiety against a pendant propionato moiety, which also increases the chelate ring
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size from a 5-membered to a 6-membered ring.352

Expanding the macrocyclic backbone by additional methylene units does not only decrease
the thermodynamic stability, but also increases the kinetic lability. This can be explained by
a higher degree of flexibility that is introduced within the macrocyclic backbone with every
additional methylene unit. This in turn accelerates the dissociation rates. Hence, Gd-DOTA
is kinetically 3 orders of magnitude more inert than Gd-TRITA and 6 orders of magnitude
more inert than Gd-TETA.354 Moreover, this example nicely illustrates that macrocyclic

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chelators do not automatically guarantee superior kinetic inertness. Other macrocyclic

systems that show rather fast dissociation kinetics were reported recently.131, 355 Increasing
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the chelate ring size of the pendent carboxylate by replacing an acetato group through a
propionato group also lowers the kinetic inertness of the system.352

Another factor that has been identified to increase the kinetic inertness of the Gd(III)-based
agent is to reduce the basicity of the ligand. In those systems, protonation of the chelator is
less favored. As a result proton-catalyzed dissociation is further hampered, which
decelerates the dissociation rate. Indeed, Gd-DOTA-tetraamide based complexes (Figure 42)
that are less basic than the parent DOTA compound were found to be considerably more
inert but at the same time they are 10-11 orders of magnitude thermodynamically less stable
than Gd-DOTA.221

Recently, a highly rigid open-chain derivative Gd-cddadpa was reported with dissociation
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rates that become comparable to inert macrocyclic complexes and sufficient thermodynamic
stability (log KGdL = 20.68, Figure 42).356 Its kinetic inertness was characterized by
studying the rate of the metal exchange reaction occurring with Cu(II) (c = 1 µM, 25 °C) at
physiological pH. Under those conditions, the dissociation half-life of Gd-cddadpa is 3
orders of magnitude higher than that of Gd-DTPA (t1/2 = 1.49 × 105 h vs. t1/2 = 202 h,
respectively) and in the range of that of Gd-DO3A (t1/2 = 2.10 × 105 h).

Ln(III) complexes of a cross-bridged cyclam derivative containing two picolinate pendant

arms show exceptional kinetic inertness, which even overcome that of Ln-DOTA complexes
by far (Figure 42).357 Under very harsh conditions, such as 2 M HCl, no sign of dissociation
has been observed for those complexes for at least 5 month. In contrast, Ln-DOTA has a
half-live of <12 hours when subjected to the same conditions. Unfortunately, the
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corresponding Eu(III) cross-bridged cyclam derivative only exhibits a hydration state of q =

0.25 – 0.35 limiting the relaxivity of this class of contrast agent. However, the correlation
between the rigidity of a molecular framework and the kinetic properties of the resulting
complex is once more highlighted with this example.

Recently, Law and coworkers reported a series of chiral, backbone substituted Gd-DOTA
derivatives, that are kinetically more inert than the parent Gd-DOTA complex.358 For
instance, Gd-L2A (SAP isomer) (Figure 42) showed almost no sign of Gd(III) release in 1 M
HCl after 8 days while t1/2 for Gd-DOTA is <25 hours when subjected to the same
conditions. Notably, these compounds afford modestly higher r1 values than Gd-DOTA (e.g.
GdL2A: r1 = 4.7 mM−1s−1, 60 MHz, human blood plasma, 37 °C) and possess very fast
water exchange rates that are in an optimal range for high field imaging (e.g. Gd-L2A: τm =
6.6 ns at 37 °C).
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A special kind of MR contrast agent are gadofullerenes that are based on carbon
nanomaterials. Fullerenes are closed up, hollow and spheroidal structures that are composed
of carbon atoms. Gd(III) can be entrapped within those carbon cages to yield paramagnetic
particles, such as Gd@C60, Gd@C82 or Gd3N@C80. The high chemical stability of
fullerenes can resist any potential metabolic cage-opening process and hence, completely
prevents the release of Gd(III) ions.359–361

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4.4 Gadolinium(III)-free alternatives

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4.4.1 Gd(III)-free small molecule relaxation agents—The Gd(III) ion is a potent

relaxation agent due to its 7 unpaired electrons and isotropic electronic ground state but
there are a few other metal ions – namely high spin Mn(II), high spin Mn(III), high spin
Fe(III), and Eu(II) that can serve as effective relaxation agents.102

The d5 Mn(II) ion is a very effective relaxation agent due to its preference for a high-spin
(S=5/2) electronic configureuration and characteristically long T1e.102 Aqueous Mn(II)
complexes typically have coordination numbers (CN) of 6 or 7.362 Whereas Gd(III)
complexes can typically accommodate both an octadentate chelator and a water co-ligand,
Mn(II) chelators cannot exceed hexadenticity and simultaneously accommodate an
exchangeable water ligand. In general the stability constants of Mn complexes are lower
compared to Gd(III) complexes utilizing comparable donor groups, and the Mn complexes
tend to be more labile. For example, log KML for [Gd(DTPA)(H2O)]2– and [Mn(EDTA)
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(H2O)]− are 22.5 and 13.9, respectively.2, 362 The dissociation half-lives of [Gd(DTPA)
(H2O)]2− and [Mn(EDTA)(H2O)]− in the presence of 10 µM challenging Zn(II) at pH 7.4 are
330 h and 0.07 h, respectively.362–363 The lower stability/inertness of Mn(II) complexes is
due to its lower charge-to-radius ratio and its absence of ligand field stabilization energy.
The poor stability and kinetic inertness of Mn(II) complexes have been the main barriers
towards the development of Mn based contrast agents that are capable of replacing Gd(III)
complexes.

A number of Mn complexes supported by various linear and macrocyclic ligands have been
evaluated as potential MRI contrast agents. (Figure 43, Table 8).362 Linear ligands like
EDTA form 7-coordinate ternary Mn complexes with a water co-ligand that are also
amongst the most thermodynamically stable Mn complexes. A number of high-relaxivity
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Mn-EDTA derivatives have been evaluated as MRI contrast agents. The benzyloxymethyl
(BOM) functionalized complexes Mn-EDTA-BOM and Mn-EDTA-BOM2 were amongst the
first reported Mn complexes of very high relaxivity. The peripheral BOM functional group
promotes high affinity interactions with HSA and Mn-EDTA-BOM and Mn-EDTA-BOM2
exhibit r1 values of 55.3 mM−1s−1 and 48.0 mM−1s−1, respectively upon HSA binding at 20
MHz and 25 °C. Mn-L1 is a high-relaxivity, serum albumin-binding Mn-EDTA derivative
analogous to MS-325. Mn-L1 was used to conspicuously detect narrowing of the carotid
artery and jugular vein in a rabbit model of arterial and venous stenosis. A hexameric Mn-
EDTA derived agent was also designed for high-relaxivity at higher (>3.0 T) field strengths.
At 4.7 T and 37 °C, a relaxivity of was 6.6 mM−1s−1 per Mn was measured for the hexamer,
whereas the monomeric EDTA-derived chelator had a relaxivity of 3.0 mM−1s−1.
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EDTA type ligands form Mn complexes that are very rapidly dissociated but replacement of
the ethylendiamine backbone with a trans-1,2-cyclohexylenediamine (trans-1,2,-
cyclohexylenediaminetetraacetic acid, CyDTA) linker increase kinetic inertness by nearly 3
orders of magnitude (Table 9).363 CyDTA also supports Mn complexes of q = 1. Recent
work evaluating the kinetic inertness of phenylenediaminetetraacetic acid (PhDTA) shows
that this ligand too provides a very kinetically inert Mn complex.356

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Although macrocyclic ligands often form complexes that are more inert that linear ligands,
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Mn complexes with hexadentate macrocylic ligands like NOTA form 6-coordinate Mn

complexes that are q = 0 and thus low-relaxivity.362 The macrocyclic chelator 1,4-DO2A has
been shown to support a q = 1 Mn(II) complex but we are unaware of any studies to evaluate
the kinetic inertness of this complex.364 However, a number of pentadentate ligands have
been reported that form q = 1 or 2 Mn(II) complexes that are less thermodynamically stable
than the complexes formed by linear and macrocyclic hexadentate chelators but may be
sufficiently inert to merit evaluation as MRI contrast agents.365–367

The chelate Mn-PyC3A was rationally designed as a potential alternative to Gd(III)-based

extracellular agents.377 The rigidifying trans-1,2-cyclohexylenediamine backbone provides
high thermodynamic stability and a high degree of kinetic inertness but leaves a vacant
coordination site for coordinately of a rapidly exchanging water co-ligand. Upon challenge
with 25 mol equiv. Zn(II) at pH 6.0, Mn-PyC3A was 20 times more inert than Gd-DTPA.
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Mn-PyC3A has a very modest affinity for blood plasma proteins and this provides a r1 of 3.8
mM−1s−1 in blood plasma at 1.4T, 37 °C, comparable to the r1 of Gd-DTPA and Gd-DOTA
measured under similar conditions (4.1 and 3.6 mM−1-s−1, respectively).59 Mn-PyC3A also
provides comparable contrast to Gd(III) contrast agents in vivo. Side-by-side comparison of
Mn-PyC3A vs. Gd-DTPA in MR angiography performed in a non-human primate model
revealed no statistically significant difference in vessel vs. adjacent muscle contrast-to-noise
ratios.378 The cyclohexylene backbone and N-pyridyl donor also provide a degree of
lipophilicity which promotes mixed renal and hepatobiliary clearance.377 Partial
hepatobiliary clearance is a favorable attribute in the context of renal impairment, as reduced
kidney function should less profoundly influence elimination of an agent with an alternate
route of elimination. Mn-PyC3A was also demonstrated to be rapidly eliminated and highly
robust against metabolic transformation or degradation.378
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High spin Mn(III) complexes such as Mn-porphyrin and Mn complexes of 1,2-phenylene

diamine derived bis-amidate ligands (Mn-PDA) have also been demonstrated as effective
MRI contrast agents.379–380 Both porphyrins and PDA type ligands form quaternary Mn(III)
complexes with two water co-ligands. The complexes are octahedral with the tetradentate
porphyrin and PDA ligands comprising the equatorial plane and the water co-ligands
occupying the apical positions. The water exchange rates of Mn-TPPS and Mn-TPMPyP
were shown be 1.4 × 107 s−1 and 1.0 × 107 s−1 at 298 K.381

The relaxivity of Mn(III)-porphyrins has been described as “anomalously” high considering

the S=2 spin state.379 The mechanisms that dictate the relaxivity of Mn(III) complexes are
largely underexplored relative to those of Gd(III) and Mn(II), but it is believed that the high
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relaxivity of Mn(III) porphyrin is at least partially due to elongation of the singly occupied
dz2 orbital which lies across the Mn(III)-OH2 bond axis. This favorable asymmetry
effectively reduces the distance between the Mn(III) spin density and the water 1H nuclei
and it has been proposed that the r6 term in equation 5 may not adequetly describe the
efficiency with which the Mn(III) ion induces 1H relaxation.379 NMRD data recorded on
Mn(III) porphyrin complexes indicate that T1e is rapid compared to Gd(III) and Mn(II)
complexes and provides significant contributions to the correlation time up to 100 MHz.
102, 379, 382 Fast electronic relaxation will limit the relaxivity gains from slow rotation. For

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example, cyclodextrin encapsulation of the Mn(III) complex Mn(III)-tpps results in only a 2-

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fold increase in r1 at 0.47 T, while the corresponding Mn(II) sister complex, Mn(II)-tpps,
showed a 6-fold increase.382

Anionic Mn(III)-porphyrin complexes have been considered as Gd(III)-free contrast agents.

For example, the tetracarboxylate functionalized complex Mn(III)-TCP was shown exhibited
comparable T1 enhancement and biodistribution to Gd-DTPA in a mouse model.383
Dimerization of a tri-sulfonated Mn(III)-porphyrin resulted in a complex that exhibited a per
Mn relaxivity that is 3-fold greater than Gd-DTPA at 3 T and 25 °C.384 This porphyrin
dimer was shown to be an effective blood pool imaging agent in a rat model.385 Mn(III)-
mesoporphyrin has been considered for use as a hepatobiliary contrast agent and was shown
to be effective for delineation of liver tumors and liver abcesses.386–387 A trisulfonated
Mn(III)-porphyrin conjugated to a 30,000 kDa dextran-derived polymer was used to
visualize lesions in a mouse model of hepatoma.388 The Mn-PDA complexes were shown to
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be cell permeable and have been further elaborated for detection of intracellular enzymes.380

High spin Fe(III) is also an effective relaxation agent. Fe(III) complexes are typically much
more stable even than Gd(III) complexes. For example, log KML for Fe-DTPA is 27.3
whereas log KML for Gd-DTPA is 22.5. The complexes Fe-DTPA (q = 0) and Fe-CDTA (q =
1) have been evaluated as extracellular contrast agents in a breast cancer xenograft mouse
model.389 The relaxivity of Fe-DTPA and Fe-CyDTA are 0.9 mm−1s−1 and 2.2 mM−1s−1,
respectively, in blood serum at 9.4 T, room temperature, compared to a value of 4.1 mM−1s
−1 for Gd-DTPA under the same conditions. Up to five-fold larger doses of the Fe(III)

complexes were required to visualize the tumors with comparable conspicuity to that
observed with Gd-DTPA. Coordinatively saturated (q = 0) complexes such as Fe-HBED and
Fe-EHPG have been evaluated as potential hepatobiliary MRI contrast agents.390–392
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High spin Fe(II) has also been considered as a contrast agent. The q = 1 Fe(II) complexes
Fe-DPTACN (DPTACN = 1,4-dipicolyl-1,4,7-triazacyclononane) and Fe-DTTACN
(DTTACN = 1,4-ditetrazoylmethyl-1,4,7-triazacyclononane) have been explored as MRI
contrast agents.393–394 A relaxivity of 0.6 mM−1s−1 was recorded for both Fe(II) complexes
at 7 T. Direct of Fe-DTTACN was shown to provide prolonged contrast enhancement in a
mouse model after direct intramuscular injection into a mouse model.393

The Eu(II) ion is isoelectronic with Gd(III) and complexes of Eu(II) have been demonstrated
to be potent relaxation agents. The Eu(II) (aq) ion has one of the fastest water exchange rates
measured.395–396 However, the Eu(II) oxidation state is disfavored relative to the
diamagnetic Eu(III) ion in the polyaminocarboxylato ligands typically utilized for Gd(III)-
based contrast agents.397–398 Eu(II)-based MRI contrast agents can be stabilized by 2.2.2
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cryptands comprised of neutral donor groups, Figure 47.399–400 The 2.2.2 cryptand cavity is
ideally suited to Ln(II) binding and the soft nature of the neutral donors accommodate the
Eu(II) oxidation state. Eu(II)-2.2.2 cryptands form ten coordinate quaternary complexes with
2 rapidly exchanging water co-ligands and thus afford high relaxivity contrast agents. The
relaxivity values and associated molecular parameters for previously reported Eu(II)
cryptand complexes are summarized in Table 10. Like Gd(III) the symmetric S electronic
ground state of Eu(II) is characterized by a T1e on the order of nanoseconds and high

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relaxivity Eu(II) complexes can be achieved by increasing τR. For example, the relaxivity of
the biphenyl appended complex Eu(II)-2.2.2-BiPh increased from 4.2 mM−1s−1 to 8.7 mM
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−1s−1 and 12.5 mM−1s−1 upon forming inclusion complexes with to β-cyclodextrin and poly-

β-cyclodextrin, respectively, and to 16.6 mM−1s−1 upon binding to HSA. Intriguingly, the
relaxivity values of Eu(II) complexes at 7 T are typically ~30-35 % greater than the values
recorded at 3 T and in this regard Eu(II) agents have received consideration for imaging
applications at field strength >3 T.401–403

Thermodynamic stability and kinetic inertness have been evaluated for a few Eu(II)-based
agents. Potentiometric titration of the Eu(II) complexes of ODDM, ODDA, and DTPA
yielded log K values ranging from 9.9-13.1.398 Similarly, log KML = 13 for Eu-2.2.2 was
determined indirectly through electrochemical measurements.399 These Eu(II) complexes
are roughly 10 orders of magnitude less stable the Gd(III)-based contrast agents but they are
remarkably inert. Evaluation of the kinetic inertness of complexes Eu-2.2.2 and Eu-2.2.2-Ph
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was evaluated under a standard set of conditions that has been applied to all of the clinical
agents (2.5 mM contrast agent, 2.5 mM ZnCl2 as challenging ion, pH 7.0 phosphate)217, 404
indicates the Eu(II) complexes are comparably inert to the macrocyclic agents such as Gd-
DOTA and Gd-HPDO3A.401

Although capable of supporting the Eu(II) oxidation state, the 2.2.2 cryptand complexes
reported to date still undergo oxidization to the corresponding Eu(III) complex under aerobic
conditions.405–407 Eu-2.2.2. is rapidly oxidized in the bloodstream, providing no positive
contrast enhancement 3 min after intravenous injection in mice.408 However, prolonged
contrast enhancement on the order of minutes following intratumoral injection of Eu-2.2.2
and for hours following intratumoral injection of Eu-2.2.2-F.406, 408 The oxidation kinetics
of EuCl2, Eu-2.2.2-F, Eu-DOTAM-Gly4 have been evaluated in the presence of the bromate
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anion and the biologically relevant oxidant glutathione disulfide.409 The data suggests that
the kinetics of Eu(III) formation reflect the overpotential between Eu(II) and chemical
oxidant. It is thus feasible that stable intravenous Eu(II) formulations could be developed by
ligand modifications to shift the oxidation potential to more positive values.

Nitroxide radicals, Figure 48, have also been considered as MRI contrast agents but are
much less effective relaxation agents than metal ion based systems.411–415 Nitroxide r1
values typically range between 0.1 – 0.5 mM−1s−1. The low relaxivity compared to
paramagnetic metal ions is due to the low spin quantum number (S = ½) as well as the fact
the nitroxide radicals do not benefit from inner sphere interactions to place the 1H into close
proximity with the paramagnet. Indeed, nitroxide radicals require stabilization from adjacent
sterically encumbering and hydrophobic functional groups.
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Nitroxide radicals are rapidly reduced to diamagnetic hydroxylamines and typically exhibit
in vivo half-lives on the order of minutes.416–420 Nitroxide reduction kinetics can be
influenced by their local chemical environment. For example, it was shown that replacing
the nitroxide adjacent geminal dimethyl groups of 3-carboxy-2,2,5,5-tetramethyl-1-
pyrrolidinyloxy (3-CP) with spirocylohexyl (chex) groups results in a 2-fold decrease in the
rate of reduction by excess ascorbate.421 Upon incorporation of chex into a
polyethyleneglycol decorated polypropylenimine dendrimers, a fraction of dendrimeric

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nitroxides become 20-fold more resistant to ascorbate reduction than the corresponding
monomer.422 These nitroxide functionalized dendrimers provide strong and persistent
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intravascular contrast enhancement that persists out to at least 1 h in a mouse model. A

recently developed nitroxide and PEG functionalized bottlebrush arm star polymer was used
to visualize subcutaneous tumors in a mouse model 20 h following intravenous injection.423

4.4.2 Iron-oxide nanoparticles—SPIONs are generally too large to extravasate into

extracellular spaces and are not excreted. The ultimate fate of SPIONs is macrophage
capture and subsequent metabolism to labile iron.424 Particle distribution is largely a
function of size.425 SPIONs of > 80 nm diameter are very rapidly captured by macrophages.
However, smaller particles can evade macrophage capture and exhibit circulatory lifetimes
that span hours to days.426 Particles > 80 nm diameter are well suited for imaging of the
reticuloendothelial system (RES). For example, the SPION formulations ferumoxide and
ferucarbotran, which are comprised of 120 – 180 nm dextran coated SPIONs and 45 – 60 nm
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carboxymethyldextran coated SPIONs, respectively, were developed for the detection of

liver lesions.49, 424, 427 Liver tissue is populated with phagocytic Kupffer cells but malignant
hepatocellular lesions are typically devoid of Kupffer cells. The strong SPION T2* effect
thus renders liver parenchyma hypointense relative to lesions containing comparatively low
populations of phagocytic cells. Similarly, the SPION T2* effect has been used to
differentiate normal from malignant, macrophage deficient lymph nodes.50, 428 SPION
opsonization also offers an effective mechanism to detect and visualize the dynamics of
inflammation.429–431 SPION enhanced MRI has been used to visualize atherosclerotic
plaque,432–434 the macrophage infiltration following stoke and myocardial infarction,435–437
as well the detection and monitoring of numerous other disease states characterized by an
acute inflammatory response.
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The smaller SPIONs are more slowly captured by the RES and are eliminated from the
bloodstream with half-lives on the order of hours to days. These smaller particles typically
have good T1 relaxivity as well and are thus ideal for contrast enhanced MR angiography.
Ferumoxytol is a SPION formulation with an FDA indication for iron replacement therapy
that occasionally receives off label use as an angiographic contrast agent.438 For example,
ferumoxytol has been used for aortic imaging,439 renal artery imaging,440 detection of
arteriovenous fistula,441 detection of deep vein thrombosis,442 and to characterize the
vasculature of brain tumors.443

Recently, a new class of iron-oxide nanoparticles small enough to clear via glomerular
filtration were introduced.444 These particles, termed exceedingly small SPIONs (ES-
SPIONs), are derived from a maghemite core which does not affect T2 relaxation as strongly
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as magnetite. The maghemite core and < 5.5 nm particle diameter provide an r2/r1 of 2.1,
which is lower than that of any other SPION. The favorable signal generating and excretion
profile of ES-SPIONs implicate these particles as potential candidates for Gd(III) free
angiography agents.

Oral SPION formulations comprised have also been developed for contrast enhanced
imaging of gastrointestinal structures. Ferumoxsil is a formulation comprised of poly-N-(2-
aminoethyl)-3-aminopropyl siloxane magnetite particles ~300 nm in diameter.54 An oral

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formulation named ferristene comprised of particles ~3500 nm in diameter has also been
developed for contrast enhanced MRI of the bowels.47
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SPIONs typically have a higher incidence of serious adverse drug reactions than Gd(III)-
based agents.445–446 SPION immunogenicity is influenced by the nature of the surface
coating and it may be possible to develop formulations of improved safety via careful tuning
of chemistry at the particle surface.447 Because SPIONs are metabolized to labile iron rather
than excreted, there are also toxicity concerns related to iron overload.448 On the other hand,
some SPIONs are highly resistant to metabolism and can persist for prolonged periods in the
liver. This can also poses a toxicity concern. For example, development of the USPION
formulation feruglose, which was designed for MR angiography, was discontinued due to
concerns over long term liver retention.47

A very large number of SPION contrast agents have been proposed and several are currently
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approved for imaging indications in the US and Europe. However, none are currently
marketed.

4.4.3 CEST agents—Although a very large number of CEST agents have been reported
over the last decade very few have been pursued with the goal of potentially replacing
relaxation agents. CEST agents are detected with poor sensitivity relative to relaxation
agents and thus larger doses are required to generate conspicuous MRI contrast. For this
reason, the vast majority of CEST agent development has focused on utilizing the versatility
of the CEST effect for specialized molecular imaging applications and are discussed in the
sections below.

Iodinated X-ray contrast agents have been evaluated as alternatives to Gd(III)-based

relaxation agents, Figure 49.449 The X-ray agents contain multiple CEST active
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exchangeable amide and alcohol protons, exhibit comparable pharmacokinetics to

commercial Gd(III) agents, and are well tolerated at the high doses required for CEST
detection. Side-by-side comparisons of contrast enhancement generated using 10 mmol/kg
of iopamidol, iohexol, and iodixanol compared to the clinically indicated 0.1 mmol/kg dose
of Gd-HPDO3A were performed in a murine tumor model.450 CEST enhancement of the
tumor expressed as (% saturation transfer) generated using the iodinated agents correlated
with Gd-HPDO3A contrast enhancement (expressed as % signal intensity increase). There
was also a strong correlation between the extravasation fractions calculated using the
iodinated contrast agents and the Gd-HPDO3A enhanced data.

A number of paramagnetic metal-ligand complexes have also been evaluated as CEST

agents. Paramagnetic ions can shift labile proton resonances by up to 500 ppm from the bulk
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water resonance and thus permits utilization of a number of proton exchanging functional
groups that would otherwise violate the Δω > kex constraint in an analogous diamagnetic
system. For example, saturation of the exchangeable Eu(III) coordinated water of Eu-DOTA
tetramide complexes has been demonstrated to provide a strong CEST effect at 7 T despite
the fact that water exchange occurs on the order of 104 - 105 s−1, which far exceeds the slow
kex required to observe the CEST effect with a diamagnetic agent, typically <103 s−1, at the
same field strength.451 CEST observation is enabled by the Eu induced >14,000 Hz shift.

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For a paramagnetic CEST agent, the benefits of a large paramagnetically shifted

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exchangeable proton resonances must be carefully balanced against the effects of

paramagnetic relaxation. For example, the amide protons of Dy-DOTAM (Figure 50)
provide a greater per exchangeable CEST effect compared to the Dy(III) bound water
because the amide protons are less effectively relaxed by the Dy(III) paramagnet.452 Thus,
the majority of newly developed paramagnetic CEST agents utilize second sphere
exchangeable protons as the spectroscopically saturable handle. Amides, alcohols, amines,
N-hydroxylamines, diazoles, and benzimidazoles have all been considered as second sphere
proton exchangers. There are elegant examples of paramagnetic CEST agents prepared with
lanthanide ions including Nd(III), Eu(III), Tb(III), Tm(III), Dy(III), Yb(III) and transition
metal ions such as Fe(II), Co(II), Cu(II) and Ni(II).199, 453–461

5. Targeted contrast agents

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Biochemically targeted agents are designed to adhere with high specificity to a molecular
target, and provide prolonged local contrast enhancement after clearance of the unbound
agent. There are myriad biochemical targets that if visible by MRI could profoundly impact
the detection, staging, prognosis, and treatment monitoring of disease, as well as elucidating
complex biology. However, developing biochemically targeted MR contrast agents is
extremely challenging. A number of considerations must be accounted for in developing a
suitable biochemically targeted agent; appropriate affinity for the target, high-specificity for
the target, rapid clearance of the unbound agent relative to washout of the target bound
agent, and high relaxivity in the target bound form.

5.1 Case study: EP-2104R, a fibrin-targeted contrast agent

We introduce this section with a detailed case study of a fibrin-targeted contrast agent to
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highlight the challenges and strategies available to develop a biochemically targeted agent.
To the best of our knowledge, the fibrin-targeted agent EP-2104R is the only biochemically-
targeted MRI contrast agent that has been used to directly detect a pathologic biomarker in
humans.462–463 Fibrin is the most highly abundant protein constituent of thrombus (clotted
blood) at up to 100s of µM, but it is not present in circulating blood or any healthy tissue.
Fibrin is formed by the actions of the enzymes thrombin and Factor XIII on fibrinogen.
Fibrinogen circulates in blood at about 7 µM and is polymerized and crosslinked into the
insoluble protein fibrin during the clotting cascade. An immediate challenge is to identify a
targeting vector that recognizes fibrin specifically over circulating fibrinogen to which it
shares great homology. Kolodziej et al. reported three families of short, cyclic peptides that
were identified by phage display to selectively bind fibrin.464 The specificity was engineered
by first screening the phage libraries against fibrinogen, removing fibrinogen binders, and
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then screening the depleted library against fibrin.

EP-2104R is comprised of 4 Gd-DOTA chelators appended to a 6 amino acid, disulfide

bridged cyclic peptide, Figure 51A. The peptide contains some unnatural amino acids found
to improve fibrin affinity.142, 464–466 The 4 Gd-DOTA chelators are included to increase the
relaxivity of the compound. It was found that conjugation of the chelates at both the C- and
N-termini resulted in compounds that were more resistant to in vivo metabolism, but at the

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same time did not substantially decrease fibrin affinity.467 The per Gd(III) relaxivity of
EP-2104R was 11.1 mM−1s−1 in buffer and increased to 24.9 mM−1s−1 when bound to fibrin
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(0.47 T, 37 °C). EP-2014R binds with little to no affinity to fibrinogen, albumin or other
blood proteins and this evidenced by the observation that relaxivity in blood plasma is
virtually unchanged from that recorded in buffer. The high fibrin-binding affinity (Kd = 1.6
for human fibrin) is reflected in the >2-fold relaxivity increase in the presence of fibrin.466

In humans, EP2104R exhibits comparable distribution to an extracellular contrast agent and

is rapidly eliminated via renal filtration with the blood signal returning to near baseline
values within 6 hours after injection.462 EP-2104R showed efficacy in detecting thrombus in
the heart, aorta, carotid arteries, deep veins, and pulmonary emoboli.462

5.2 Challenges and New Frontiers

MR imaging of fibrin provides but one illustrative example of the potential impact of a
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biochemically specific MRI contrast agent. Successful translation of EP-2104R was the
culmination of years of dedicated research and development effort and provides several
valuable lessons for the design of a biochemically targeted contrast agent. In this section we
will review prior accounts describing the design and application of MRI contrast agents
targeting various biomarkers in order to illustrate the challenges and innovative solutions
toward this new frontier in contrast enhanced MRI. To extend this work to other targets, a
number of technical challenges must be addressed. Identifying targeting vectors to bind to a
specific molecular target with high affinity and high specificity is itself very difficult.
Appending a contrast generating moiety to the targeting vector in a manner that does not
compromise target affinity adds another layer of complication.

A third challenge is that micromolar concentrations of metal ion are required to generate
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detectable contrast with T1- or T2-weighted imaging sequences. Few biochemical targets are
present at high enough concentration to be imaged with a targeted agent containing only a
single metal ion. However, very innovative strategies have been explored to amplify contrast
upon encountering its biochemical target. These strategies can be broadly divided into 4
general approaches, each with their own strengths and weaknesses: (1) increase the
contrasting payload by conjugating multiple relaxation agents to the targeting vector. This
strategy was successfully pursued in the EP-2104R example with 4 chelates per peptide.
Such agents are chemically well defined molecules that can be reproducibly synthesized.
They are relatively small molecules which allows them to rapidly distribute in the body, bind
to the target, and see the unbound agent be quickly eliminated from the body. The small size
also typically results in ultimately complete elimination from the body. Drawbacks include
the increasing complexity of the molecule and its synthesis, as well as the dearth of relevant
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molecular targets at high enough concentration. (2) Take advantage of an endogenous

mechanism such as receptor mediated internalization to accumulate the targeted agent at MR
visible concentrations. This is in principle a very attractive technique however the
internalization process must be very efficient to accumulate the micromolar concentrations
required for detection. (3) Utilize nanoparticles that bring hundreds or thousands of metal
ions to increase the detection sensitivity into the low nanomolar range. (4) Use nanoparticle
approaches to deliver a very high payload of non-endogenous (i.e. background free)

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magnetically resonant nuclei such a F-19. Both nanoparticle approaches are attractive
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solutions to overcoming sensitivity but bring challenges in terms of reproducibility,

pharmacokinetics, and elimination. For clinical translation, the agent must be well defined
and there are obvious challenges in controlling the distribution of nanoparticles both with
respect to their size and their surface derivatization, although these can be overcome.
Nanoparticles typically distribute in the blood and are captured by macrophages in the liver,
spleen, and lymph nodes. Tumor accumulation can occur either via capture in tumor
associated macrophages or via the enhanced permeability of many tumors, but these process
take several hours. In general nanoparticles are not completely eliminated from the body and
this can also create concerns for clinical translation.

5.3 Fibrin
As discussed above, fibrin is the main protein constituent of clotted blood and also present in
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the tumor microenvironment making it a useful protein target in the molecular imaging of
thrombosis,462, 468 cancer,469 and other disease states that possess a coagulative component.
In addition to the human EP-2104R imaging described above a few additional fibrin
targeting contrast agents merit note. A Mn-based fibrin seeking contrast agent, Mn-FBP, was
also reported, Figure 52.377 This agent uses the same peptide as EP-2104R but the 4 Gd-
DOTA chelators are replaced with 4 Mn-PyC3A moieties. Mn-FBP demonstrated equivalent
fibrin affinity to EP-2104R in vitro and provided equivalent contrast enhancement of arterial
thrombi in rats. The high-payload strategy was extended even further in a liposomal fibrin-
targeted agents, where fibrin antibody was conjuguated to liposome comprised of self-
assembled amphiphilic Gd(III) complexes.470–472 The direct nuclear detection strategy has
been pursued by conjugating 19F perfluorocarbon nanoemulsions to anti-plasmin peptide
fragments which become chemically crosslinked to fibrin.473
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5.4 Serum albumin

Albumin is a serum protein that is present at ~660 µM and thus represents an abundant target
for intravascular contrast agents. Small molecule agents with high affinity for albumin will
confine largely to the blood pool (the 66 kDa protein does not extravasate) and provide
prolonged and strong intravascular contrast. For example, MS-325 is an FDA approved
albumin targeting agent that non-covalently binds to the albumin drug binding site II with Kd
= 85 µM.17, 139, 474–475 MS-325 binding is promoted by a 4,4-diphenylcyclohexyl moiety
connected to the Gd-DPTA chelate via a phosphodiester linkage.16, 476 The µM binding of
MS-325 affinity ensures that a small fraction (10-20%) of MS-325 remains unbound at all
times, and this unbound fraction can be cleared via glomerular filtration. Le Chatelier’s
principle ensures that this unbound fraction is constantly reestablished. Albumin is also
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present in the lymphatic system, and MS-325 has been used to detect metastatic invasion of
lymph nodes.477 In certain pathologies albumin can leak out of the blood vessels into the
interstitial space. Montesi et al. showed that patients with idiopathic pulmonary fibrosis had
extensive vascular leak in their lungs, readily detected by MS-325 enhanced MRI.478 A Mn-
EDTA analogue of MS-325 has also been reported.331

A number of additional albumin targeting functionalities also have been appended to

relaxation agents for intravascular imaging. For example, the Gd-DTPA based agents

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B22956/1, which has been extensively evaluated as an angiographic contrast agent, utilizes
3-deoxycholic acid as an albumin targeting group.23–26, 479–482 A biological function of
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serum albumin is to transport lipopilic anions. The high concentration of serum albumin and
its ability to promiscuously binding many classes of compounds makes it possible to
generate a large number of albumin-targeted agents.483–485 Additional examples of albumin
binding MRI contrast agents have been reported,333, 476, 486–489 but there are too many to
acknowledge even for this extensive review.

5.5 Type I Collagen

Type I collagen is the most abundant protein constituent of connective tissue. In fibrosis, or
organ scarring, collagen is overexpressed and is present at 10s of µM concentration. Fibrosis
occurs in a number of major diseases such as myocardial infarction, heart failure, atrial
fibrillation, hepatitis and cirrhosis, diabetic nephropathy, pulmonary fibrosis, inflammatory
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bowel disease, and a number of cancers. The type I collagen-targeting contrast agents
EP-3533,490 EP-3600,491 and CM-101492 were developed by conjugating Gd-DTPA or Gd-
DOTA chelators to peptides that were identified by high-throughput screening methods to
bind to type I collagen with high specificity, Figure 53. These collagen-targeting agents all
capitalize on the increased payload strategy, with 3 Gd(III) chelates per peptide in each
compound. EP-3533 has been used to stage liver fibrosis in mice and rats and also to
monitor response to drug therapy.493–494 It was also shown to be effective to detect and
quantify the extent of myocardial infarction,495 to stage tumor associated fibrosis,496 and to
stage pulmonary fibrosis in mouse models.497 EP-3600 was used to image perfusion defects
in a porcine model,491 and CM-101 was reported for imaging liver fibrosis in rat and mouse
models.492

Figure 54 compares liver imaging data acquired in healthy rats and rats experiencing liver
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fibrosis resulting from bile duct ligation (BDL). Figure 54A compares the time course of
liver-to-muscle contrast to noise ratio (CNR) observed in control (sham surgery) and BDL
rats following injection of equal doses CM-101. CM-101 is rapidly distributed and
eliminated and liver contrast in control rats is accordingly diminished to near baseline levels
within 15 min of injection. In the fibrotic liver however, CM-101 adheres to collagen and
provides high liver CNR at delayed time points after washout of the unbound agent. Figures
54B-C further illustrate this concept by comparing liver CNR integrated over 0-30 min for
the sham surgery and BDL rats and liver CNR between sham surgery and BDL rats 10 min
after CM-101 injection. Figure 54D compares T1-weighted MR images of the sham and
BDL (fibrotic) rats prior to and 15 min after CM-101 injection.

5.6 Fibrogenesis
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Fibrogenesis, or the active deposition of scar tissue, is characterized by high levels of

reactive aldehydes which are generated as part of a lysyl oxidase (LOX) mediated collagen
crosslinking process. LOX catalyzes oxidative deamination of lysine side chains to form
allysine, which goes on to participate in condensation reactions with nearby lysine and
allysine residues, resulting in crosslinking between collagen strands. Although allysine is
reactive, the transiently generated aldehyde is present in sufficiently abundant concentrations
for MR detection using an aldehyde-targeting imaging probe.498 Allysine has been targeted

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using Gd(III)-based agents functionalized with hydrazide and oxyamine targeting moieties,
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which attach to allysine via reversible formation of hydrazone and oxime bonds. The
hydrazide containing, aldehyde targeting agent Gd-Hyd, Figure 55A, was used to measure
the natural history of bleomycin induced fibrogenesis in the lungs of mice, as well as the
response to treatment with a LOX inhibitor.499 Gd-Hyd was also capable of monitoring liver
fibrogenesis in a CCl4 mouse model.

Similarly, Gd-OA, Figure 55A, with a pendant oxyamine moiety was used to detect
bleomycin-induced pulmonary fibrogenesis in mice.500 Figures 55B-D compare lung
imaging data and lung allysine content in normal, naïve mice vs. mice experiencing
pulmonary fibrogenesis as a result of bleomycin injury. Figure 55B compares the change in
lung-to-adjacent muscle signal intensity ratio following contrast agent injection, and
demonstrates strong lung enhancement in the bleomycin treated mice. The MRI
measurement reflects ex vivo quantification of tissue allysine content, Figure 55C, which is
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present at high micromolar concentrations in the injury lung. Contrast enhancement is

clearly evident in the lungs of the bleomycin injured mice as compared to naïve mice, Figure
55D.

The aldehyde-targeting mechanism of both agents was confirmed using the control agents
Gd-DiMe and Gd-OX, Figure 55A, which exhibit comparable pharmacokinetics and
relaxivity but are incapable of forming hydrazone and oxime bonds, respectively. These
control agents provide no contrast enhancement in tissues experiencing active fibrosis.

5.7 Fibronectin
Aggressively metastatic tumors are typically characterized by interstitial coagulation and
thus proteins such as fibrin and fibronectin represent promising prognostic molecular
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imaging targets. In addition to the fibrin-binding peptides discussed above, other high-
throughput screens have yielded low-molecular weight peptides that bind to an epitope
formed by the fibrin-fibronectin complex with good affinity.501 Two small fibrin-fibronectin
targeting peptides CGLIIQKNEC (CLT1) and CREKA have been utilized for molecular
imaging of cancer, Figure 56A. CLT1 conjugated to Gd-DTPA via an amide linkage at the
peptide N-terminus was shown to provide strong delayed enhancement of human colon
carcinoma xenografts in mice long after probe washout.502–503 High payload fibronectin-
targeting contrast agents have also been developed by appending a tetrameric Gd(III)-
complex to the N-terminus, Figure 56B, or a trimeric Gd(III)-complex to the cysteine-S,
Figure 56C, of the CREKA peptide. These higher relaxivity agents have been used to
effectively detect orthotopic prostate and breast tumors in mouse models as well as
micrometastases <0.5 mm in diameter, which are difficult to detect using conventional
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extracellular contrast agents.504–506 The fibronectin selectivity of the CREKA-based agent

was further evidenced by comparing localization of a fluorescent peptide with a fibronectin
specific immunohistochemical stain using fluorescence microscopy ex vivo. Imaging
performed with an otherwise identical agent built from the scrambled CERAK peptide (non-
binding control) does not provide delayed tumor enhancement.

Fibronectin-targeted “platelet-mimetic” SPIONs have been developed that promote further

particle accumulation around particles bound to vessel wall.503 These fibronectin-targeting

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SPIONs accumulate in the vessel walls of proliferating tumors and were used for
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fluorescence-based imaging of human breast cancer xenografts in mice. The fibronectin-

targeting SPIONs did not localize to the same tumor phenotype in fibrinogen knockout mice
which are incapable of expressing the fibrin-fibronectin complex, further confirming the
mechanism of tumor localization. These particle have not yet been applied to MR imaging
studies, however, but one could expect the “platelet-mimetic” mechanism of SPION
accumulation to provide very strong contrast enhancement in similar models.

5.8 Elastin
Elastin is an abundant protein component of load bearing tissues and levels of this protein
are largely increased as part of the compensatory response following injury. Molecular
imaging of elastin was performed with the elastin-seeking contrast agent, Gd-ESMA, Figure
57A, which comprises an elastin-binding small molecule conjugated to Gd-DTPA via an
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amide linkage involving one of the DTPA carboxylates. Gd-ESMA was used to monitor
changes in elastin composition in the vessel wall of arterial aneurysm,507 vascular
remodeling following atherosclerotic plaque disruption,508 and changes in the extracellular
matrix of cardiac tissue following myocardial infarction.509–510

The images in Figure 57B-G show the abdominal aortas of a mouse bearing a
pharmacologically-induced abdominal aortic aneurysm (B-D) and a placebo treated mouse
(E-G). The red lines in the angiograms shown in Figures 57B and 57E mark the axial cross
sections of the vessels depicted in images Figure 57C-D and Figure 57F-G, respectively. The
cross sectional images were collected with no contrast enhancement and several minutes
after Gd-ESMA injection. Elastin is present in all aortas and the vessel wall in the placebo
treated mouse is slightly enhanced but the compensatory remodeling of the vessel wall at the
site of the aneurysm results in increased elastin content in the vessel wall, which is very
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strongly contrast enhanced.

5.9 Myelin
A number of neurodegenerative disorders are characterized by disruption to the myelin
sheath surrounding the axons of white matter nerve cells. A myelin-seeking contrast agent
was developed by appending a Gd(III) complex to a 3-(4-aminophenyl)-2H-chromen-2-one,
termed Case Myelin Compound, or CMC, Figure 58.511–512 CMC was identified from a
screen of myelin adhering coumarins as the molecule with greatest affinity to myelin.
Myelin localization of CMC was confirmed by MR imaging of contrast agent incubated
slices of excised mouse brain in vitro, and comparing the patterns of coumarin fluorescence
to myelin specific immunohistochemical staining.512 CMC was also shown to bind myelin
sheaths in vivo following intravenous injection.513 Intracranially administered CMC enabled
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MR visualization of pharmacologically induced demyelination in a rat model in vivo.511

5.10 Amyloid-β peptides

Insoluble amyloid-β (Aβ) peptides are an important biomarker for detection of Alzheimer’s
Disease. Aβ is a particularly challenging target because most contrast agents are incapable
of penetrating the intact blood-brain barrier. However, innovative chemistry has yielded a
handful of contrast agents that can cross the blood-brain barrier and provide enhancement at

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the site of Aβ deposits. For example, an Aβ fragment containing amino acids 1-40 (Aβ1-40)
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conjugated to Gd-DTPA and putrescine at the N- and C-termini, respectively, was shown to
penetrate the blood brain barrier and adhere to Aβ depositions.514 Incorporation of the
polyamine putrescine confers blood brain barrier permeability. This effect had been
previously demonstrated with putrescine conjugated proteins.515 The Aβ1-40 aggregates
with the insoluble Aβ. In vivo imaging was not performed with this agent but ex vivo brain
slices from an Alzheimer’s Disease mouse model, excised after injection of the agent, were
imaged with MRI. The T1 enhancement patterns of the brain slices localized well with
Thioflavin S staining for insoluble Aβ. A similar strategy has been taken to access Aβ-
deposits with USPIONs. The USPIONs were decorated with a Aβ1-42 as the targeting
vector and polyethylene glyocol in order to increase membrane permeability.516 The Aβ-
seeking nanoparticles provided strongly enhanced T2* contrast 4h after injection in the
brains of Alzheimer’s bearing mice compared to wild type mice. In another study, an Aβ-
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targeted agent comprising Gd-DTPA coupled to N-terminus of a K6Aβ1-30 peptide was co-
injected with mannitol in order to relax the blood-brain barrier in a mouse model.517 A
series of Gd-DOTA complexes conjugated to Pittsburgh Compound B, a well-established
PET tracer for imaging Aβ deposits, were shown to bind to Aβ1-40 in vitro.518–519

Alzheimer’s disease can also be accompanied by neuro-inflammation that can significantly

compromise endothelial integrity. Aβ-targeted agents comprising SPIONs coupled to Aβ
protein precursor antibodies (Anti-AβPP) have been shown to access Aβ depositions via this
endogenous mechanism of blood-brain barrier breakdown. Imaging was performed ex vivo
on brain slices and correlated to Aβ histologic staining after administration of the Anti-
AβPP nanoparticles to Alzheimer’s bearing mice.520

5.11 Organic Anion Transporting Peptides

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Organic anion transporting peptides (OATPs) are highly expressed on the surface of
hepatocytes. OATP mediated internalization has been utilized to develop liver targeting
agents such as the commercially available Gd-BOPTA and Gd-EOB-DTPA.521–523 Although
these clinical agents are not typically thought of as biochemically-targeted contrast agents,
the strong liver contrast observed following injection is the result of receptor mediated
pooling of the agent in hepatocytes. OATPs are highly abundant on the surface of normal
hepatocytes but are under-expressed on the surface of malignant cells and thus OATP-
targeted agents are used to render malignant hepatocellular lesions conspicuously
hypointense relative to healthy liver parenchyma. Hepatocellular phase images are typically
acquired 20 – 40 min after injection in humans. OATP agents are also used in the differential
diagnosis of hepatocellular lesions from benign, dense clusters of normal hepatocytes
termed focal nodular hyperplasia (FNH). FNH typically exhibit avid arterial phase
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enhancement similar to many malignant lesions but unlike malignant lesions FNH are
strongly enhanced by OATP-targeting agents during the delayed hepatocellular phase.524
Recently, a Mn based hepatocyte seeking agent was developed by conjugating Mn-EDTA to
a benzothiazole aniline (BTA) moiety.525 Hepatocyte uptake presumably occurs via OATP
mediated transport, but the mechanism has not yet been formally interrogated. A 0.05
mmol/kg dose of Mn-EDTA-BTA was used to visualize liver tumors in a mouse model with

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high contrast. The liver tumors were hypointense relative to the strongly enhanced liver
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parenchyma, consistent with previously developed OATP targeting agents.

Patrick et al. utilized the tremendous accumulating power of OATP as a gene reporter. They
expressed the OATP receptor into cancer cells and implanted them into mice. Using Gd-
EOB-DTPA for detection, they showed that cells expressing the reporter showed readily
reversible, intense, and positive contrast (up to 7.8-fold signal enhancement) in T1-weighted
magnetic resonance images acquired in vivo.526

5.12 Proliferating cells

Increased glucose metabolism is a hallmark feature of proliferating tumors. Malignant cells
have high glycolytic activity compared to non-cancerous cells and typically overexpress
insulin independent glucose transporters. Indeed, this is the physiologic basis of clinical
[18F]fluorodeoxyglucose (FDG) PET scans to detect malignancies. Innovative studies have
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demonstrated that analogous imaging can be performed using glucose and glucose
derivatives as DiaCEST MRI contrast agents, making use of the chemical exchange of the
hydroxyl protons. This targeted imaging is another example of receptor mediated
concentration of contrast agent. The efficacy of this approach has been demonstrated in both
animal models and human patients.527–530 It was recently shown that glucose can be used to
generate strong contrast enhancement in newly diagnosed, untreated glioblastomas using
chemical exchange spin locking scans – which are analogous to CEST scans but more
clinically robust and sensitive to hydroxyl proton exchange.531 Figure 59B shows glucose
enhanced visualization of the proliferative microenvironment in a human glioblastoma
patient.

The hallmark leaky vasculature of solid tumors enables targeting cancer cells and
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extracellular proteins with macromolecular agents, although the pharmacokinetics of these

agents are markedly slower and imaging must be performed hours later than would typically
be done using biochemically-targeted small molecule agents. The high metabolic demands
of proliferating cells lead to increased consumption of nutrients such as iron and this
increased nutrient demand has been exploited for targeted molecular imaging (i.e. receptor
mediated accumulation). For example, transferrin-SPION conjugates have been used to
image mammary carcinoma in a rat model.532

The protein nucleolin is highly overexpressed on the surface of continuously proliferating

cells and plays a critical role in promoting anti-apoptotic activity. A proliferation-seeking
contrast agent was developed by derivatizing SPIONs with AS1411 aptamers, which bind
surface expressed nucleolin with high affinity. This agent was shown to selectively adhere to
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cells with surface expressed nucleolin and was successfully used to delineate proliferating
tumors via T2-weighted scans in a mouse model.533

Angiogenesis is another hallmark feature of the proliferative microenvironment as the

formation of new blood vessels enables continued nourishment of the proliferating cells and
an avenue for metastasis. In this regard, contrast agents have been developed to target the
ανβ3 integrin receptors highly expressed on endothelial cells participating in angiogenesis.
An ανβ3-seeking contrast agent was developed by linking a biotinylated ανβ3 –specific

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monoclonal antibody, DM101, to a biotin-coated Gd(III)-labelled perfluorocarbon

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nanoparticle. Avidin, which binds biotin with sub-pM affinity was used to link the
biotinylated particle and antibody.534 The antibody based contrast agent provided 25%
signal enhancement at the locus of corneal angiogenesis in a rabbit model 4h following
intravenous injection, whereas no contrast enhancement was observed at the same time point
using the paramagnetic nanoparticle alone, the corresponding non-binding isotype control,
or administration of the ανβ3-seeking agent following pre-injection receptor blocking using
a competing substrate. Numerous ανβ3-seeking agents have been developed using the cyclic
tripeptide RGD as the integrin-binding moiety. For example, RGD functionalized SPIONs
have been used to detect ανβ positive vasculature.535–536 One study demonstrated that
relative SPION induced change in intratumoral T2 was shown to correlate with
immunohistochemically determined ανβ3 per unit area coverage.536 Polymeric and
liposomal Gd(III)-based agents and CEST reporters have also been developed as high-
payload integrin-targeting agents.537–538
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High-affinity folate receptors (HFR) are overexpressed on several tumor phenotypes

originating from endothelial cells. A HFR targeting contrast agent was developed by
conjugating DOTA to folate via bis(aminoethyl)glycol linker which formed amide bonds
with both DOTA and folate carboxylates, Figure 60. This small molecule agent was
successfully used to provide delayed enhancement of a HFR-positive human ovarian cancer
xenograft in a mouse model for hours after injection of Gd-DOTA-folate, whereas no
delayed enhancement was observed in control mice bearing HFR-negative xenografts.539 A
high relaxivity, folate-targeting Gd(III)-based agent of undisclosed structure has also been
developed. This agent, termed P866, was used to provide strong contrast enhancement of
HFR positive tumors in mice for hours after injection.540–541 Contrast enhancement due to
HFR binding was confirmed via imaging with a non-HFR binding control agent and by
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imaging using P866 under conditions where HFR was blocked by injection of free folate.
Folate functionalized SPIONs have also been used for visualization of HRF-positive tumors.
541 HFR has also been targeted with high-payload agent comprising a 4th generation

PAMAM dendrimer functionalized with both folate and Gd-DTPA. A typical dendrimer was
functionalized with 3-5 folate molecules but dozens of Gd-DTPA complexes. These folate
targeting agent were shown to provide strong contrast enhancement of HFR-positive ovarian
cancer xenografts in mice 24h after injection.542 Although no imaging was performed using
a non-targeted control dendrimer, enhancement due to a folate-targeting mechanism was
retrospectively confirmed by comparing Gd(III) retention in tumors using targeted and non-
targeted dendrimers labelled with 153Gd(III) by gamma counting methods.543 A number of
groups have also developed HFR-targeted agents using polymers and liposomes to deliver a
high-payload of Gd(III)-based contrast agent.544–547
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5.13 Inflammation
Inflammation has been imaged using agents that target the cell adhesion molecules that play
a critical role in the early stages of inflammation by recruiting circulating leukocytes to the
site of injury. A “leukocyte-mimetic” SPION agent was developed by coating 1 µm diameter
SPIONs with antibodies against the cell adhesion molecules P-selectin and vascular cell
adhesion molecule-1 (VCAM-1).548 These particles were used to image endothelial

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activation during the onset of atherosclerosis in the aorta of mice. MR contrast correlated
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with lesion macrophage content. Flow cytometry experiments confirmed that these
“leukocyte-mimetic” agents adhered to activated endothelial cells and were not internalized
by phagocytic macrophages. A SPION functionalized with P-selectin antibodies was also
used to monitor the dynamics of spinal cord endothelial activation in mouse models of
chronic and relapsing multiple sclerosis.549 The cell adhesion molecule E-selectin has been
targeted using a dextran-coated SPION functionalized with a small molecule mimic of the
E-selectin targeting polysaccharide sialyl Lewisx (sLex) was used to image liver endothelial
cell activation in a mouse model of hepatitis.550 This small molecule sLex mimic was also
conjugated to Gd-DTPA type contrast agents.551 The resultant agent, Gd-DTPA-B(SLex)A
Figure 61, was shown to exhibit a ~67% increase in blood-half life in a rat model of
fulminant hepatitis (30 min vs 49 min in healthy vs. hepatitis rats, respectively) which was
attributed to interactions with endothelial cell adhesion molecules in the diseased rats.552 In
mice, Gd-DTPA-B(SLex)A, was shown to provide strongly enhanced intravascular contrast
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out to 1 h after injection, whereas blood signal returned to near baseline within minutes after
injection of Gd-DTPA.552

The inflammatory microenvironment is rich with phagocytic macrophages. Phagocytosis has

been pursued by a number of groups as an endogenous mechanism to concentrate
macromolecular contrast agents such as SPIONs,430, 553 Gd(III)-loaded liposomes,554 and
19F perfluorocarbon nano-emulsions555–557 for targeted imaging of inflammation.

5.14 Apoptosis
Apoptosis is programmed cell death in response to cell stress or cell signals but dysregulated
apoptosis is often encountered in the proliferative microenvironment of solid tumors.
Apoptotic cells are characterized by highly negatively charged phosphatidylserine rich cell
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membrane and this unique cell surface has been used as a targeting mechanism. A high
payload, protein-based apoptosis-targeting agent was developed by conjugated Gd-DTPA to
the surface exposed lysines of glutathione-S-transferase-C2A fusion protein, which binds to
the phosphatidylserine rich surface of apoptotic cells with high affinity.558 This protein
based agent was shown to bind selectively to apoptotic and necrotic cells in vitro and was
used to image apoptotic cells in a mouse model of lymphoma. Another way to target
apoptosis with high payload is to use Gd(III)-chelate loaded liposomes conjugated to Anexin
5, which also has high affinity for apoptotic cells.559 These liposomal agents have been used
for targeted imaging of apoptosis in atherosclerotic plaque in mice.560 Anexin 5
functionalized SPIONs have also been used to image cardiomyocyte apoptosis in a mouse
model of myocardial ischemia.561
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5.15 Necrosis
There are high concentrations of extracellular DNA in acutely necrotic tissue, as
macromolecular detritus requires hours to days to clear from the newly necrotic region. In
this regard, an extracellular DNA-binding agent was developed for molecular imaging of
necrosis.562 The agent DNA-binding agent, termed Gd-TO, Figure 62, is comprised of Gd-
DOTA coupled to the DNA intercalator thiazole orange. Gd-TO was used for molecular
imaging of acute necrosis following myocardial infarction in a mouse model. The dynamics

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of extracellular DNA clearance were monitored by Gd-TO enhanced imaging at various time
points between 0 to 72h after myocardial infarction.562
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6. Activatable contrast agents

Besides direct targeting, molecular MRI can also exploit changes in relaxivity or chemical
exchange to detect specific biological processes or pathologies. Ion flux, pH, enzymatic
activity, chemical potential (redox), and temperature are all features of the
microenvironment that can be altered in disease processes. Activatable contrast agents,
sometimes called responsive or ‘smart’ contrast agents, exhibit altered relaxivity or altered
CEST effect in response to a stimulus. Unlike targeted agents which accumulate at a specific
site because of protein binding or cellular internalization, activatable agents produce signal
change because they themselves are chemically altered. Activatable contrast agents that are
capable of revealing biochemical or physiologic abnormalities may offer unique insights into
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the human body and enable the earlier and more precise diagnosis of specific diseases, as
well as monitoring the treatment of these disorders.

This section provides an overview of the chemistry, properties and applications of

activatable para- and diamagnetic contrast agents. The probes discussed here can be viewed
as MR sensors of physiological events whose relaxivity or CEST effect depends on the
absence/presence of the biological stimulus of interest.

As outlined in section 3.3, the T1 relaxivity of a paramagnetic metal complex is primarily

determined by three factors: q, the number of water molecules in the inner coordination
sphere, τm, the residence lifetime of these inner-sphere water molecules, and τR, the
rotational correlation time of the molecule. Thus, for the development of a biochemically
responsive contrast agent, one or more of these molecular factors must be changed by a
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biochemical stimulus. In the ideal case, the agent features an “off” state associated with low
relaxivity in the absence of the biochemical trigger and an “on” state associated with high
relaxivity in the presence of the trigger. Similarly, superparamagnetic nanoparticles can
exhibit activatable T2* change based on aggregation of the particles. CEST agents can be
made responsive by altering the chemical exchange rate or the chemical shift of the
exchangeable hydrogen in response to a stimulus, which causes the CEST effect to appear,
disappear or change in magnitude.

6.1 Case study: A Gd(III)-based β-galactosidase sensor

Pioneering work in the development of activatable Gd(III)-based contrast agents was led by
Meade and coworkers with their studies on agents responsive to the enzyme β-galactosidase.
563 β-galactosidase catalyzes the hydrolysis of β-galactosides and is commonly used in
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molecular biology as a reporter marker to monitor gene expression.564–565 The Meade group
sought to make a MR probe that could report on β-galactosidase function and gene
expression. The initial sensor (Figure 63A) consisted of a Gd-DO3A chelate coupled to a
galactopyranose moiety that acts as a lid for the access of water to the inner sphere
coordination cage. Enzymatic cleavage of the glucosidic linkage irreversibly frees a
coordination site to yield a Gd-HPDO3A-like complex with an increased number of inner-
sphere water molecules resulting in higher relaxivity. Using lifetime luminescence

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measurements on the Tb analog, the authors estimated that the hydration state of the
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complex changes from 0.7 to 1.2 upon cleavage of the sugar group. The relaxivity change
was modest but could be improved by modifying the complex to introduce an additional α-
methyl group within the molecular scaffold as depicted in Figure 63B-C.566 This
modification appears to reduce the relaxivity of the uncleaved complex with the
galactopyranose moiety intact, presumably by limiting water access to the gadolinium(III)
center. Using this second generation probe, β-galactosidase activity could be detected in
living Xenopus laevis embryos (Figure 63D). Two embryos were injected with the
activatable contrast agent, while one embryo was additionally injected with β-galactosidase
mRNA. The MR signal intensity was 45 – 65 % greater within the embryo that was treated
with β-galactosidase mRNA, thereby demonstrating the detection of β-galactosidase activity.

This study demonstrates the power of activatable agents to detect biochemical events,
notably enzymatic activity. In this case, 55 M water signal was imaged and this signal was
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augmented by 0.5 mM contrast agent which in turn was augmented by a 4 µM enzyme

concentration. Thus activatable agents offer an avenue to detect low concentration molecular
events, despite the sensitivity limitations of MRI. The study also points to some requisites
for activatable agents. The difference in relaxivity (or CEST effect) between the “off” and
“on” state should be large to detect meaningful change. Additionally, the reaction to activate
the agent must be very fast; for enzyme sensing, the kinetics must be fast enough to turnover
micromolar concentrations of agents in a short period of time.

6.2 Challenges and New Frontiers

Activatable agents have the same primary challenge as all the other MR contrast agents
described in this review: detection sensitivity. For relaxation agents, micromolar
concentrations of metal ion are required and millimolar concentrations are needed for CEST.
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Direct detection typically requires even higher concentrations. Regardless of the mechanism
of activation and the magnitude of change in signal generating property (relaxivity, chemical
exchange rate), the activatable agent can only be effective if it is present at concentrations
above its detection threshold.567

Another consideration is whether the activation is reversible or irreversible. Reversible

responsive MRI contrast agents return to their original state when they no longer interact
with their specific biomarker. This makes them especially suitable for monitoring rapidly
changing physiological conditions, e.g. ion flux. But in this case the MR detection
sensitivity is target-limited, which means that the biomarker of interest has to be present in
relatively high concentrations to be detectable with a reversible activatable probe.
Irreversible responsive agents do not return to their original state in the absence of their
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specific biomarker. With irreversible responsive agents, signal change is one way and
oscillations in biomarker concentration cannot be detected. On the other hand, the complete
conversion to the activated state can greatly amplify detection sensitivity.

There are two main approaches to enhance dynamic range of both reversible and irreversible
responsive agents. One strategy is to maximize the signal change between the inactivate and
the activated agent. This can be done by optimizing the relaxivity (or CEST effect) of the
activated agent or by suppressing the relaxivity of the inactive form, or a combination of

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both. Again, one must keep in mind that the overall detection threshold must be reached, so
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for relaxation agents the relaxivity of the activated agent must be high enough to render the
agent detectable at physiologically achievable concentrations. In another strategy the
responsive agent is not only “turned-on” in the presence of a particular biological stimulus
but additionally retained and thereby accumulated at the target. This approach can be seen as
a combination of targeted and activatable probes and will be further discussed in section 7.

Another question to address is whether one wants to simply detect the presence of a
biomarker, e.g. the presence of a specific enzyme, or whether one wants to quantify a
biomarker, e.g. measure pH. Quantification presents a particular challenge because the MR
signal depends not only on the response to the biochemical stimulus (relaxivity change) but
also on the concentration of the imaging probe. Even for qualitative detection, it is necessary
to insure that the signal change observed is a result of activation and not just pooling of the
inactive agent. Three general methods have been employed to address this problem: (1) the
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utilization of ratiometric methods where the measured effect is independent of

concentration; (2) successive injections of responsive and non-responsive agents with similar
structure and pharmacokinetics where the signal change of the non-responsive agent is used
to estimate the concentration; or (3) utilization of a bimodal agent and simultaneous bimodal
imaging, where one image modality (PET or SPECT) is used to quantify the agent and this
is used to deconvolute the MR signal and quantify the degree of activation. These strategies
will be discussed on the basis of exemplary examples.

As seen below, a large number of activatable agents have been reported. However unlike
with targeted agents, there have been relatively few successful examples of activatable
agents being deployed in vivo. On the other hand, the ability to image and quantify
enzymatic activity, ion flux, redox status, pH, etc deep within tissue with submillimeter
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resolution would have a tremendous impact on our understanding of biology and

pathobiology. The development of truly functional activatable agents remains a worthy goal.

Excellent reviews of activatable MR contrast agents have been published in recent years.
185, 567–572 In this section, our primary goal is to provide an overview of the different

strategies and creative ideas that have been employed in the design of activatable MR
contrast agents. Due to the large number of examples in the literature, this section is not
meant to be comprehensive. We regret the omission of interesting studies for the sake of
conciseness.

6.3 pH-responsive molecular imaging agents

Decreased extracellular pH values are associated with cancer and various ischemic diseases.
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pH quantification could be a useful biomarker for early identification of disease or to

monitor treatment efficacy.573 Hence, the development of smart MR contrast agents, which
show pH-dependent relaxivity profiles or CEST effects has been the subject of intensive
research for the past two decades.

6.3.1 pH-responsive Gd(III) based contrast agents—An early and effective

example of a pH-responsive agent was Gd-DOTA-4AmP (Figure 64A) reported by Sherry
and coworkers.574–575 This complex is a DOTA derivative where the carboxylate groups

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have been derivatized as amides with pendant phosphonate groups. Inner-sphere water
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exchange is extremely slow for this complex, but exchange of protons of the bound water
molecule with bulk water is fast and is catalyzed by pendant phosphonates. Protonation of
the phosphonate groups, which have pKa values in the physiological range, alters this
prototropic exchange rate and also alters second-sphere relaxivity. The relaxivity is 51 %
higher at pH 6 compared to pH of 9.5. The magnitude of the pH dependent relaxivity effect
can be increased by slowing the overall tumbling rate of the molecule. Coupling of this
agent to the macromolecule G5-PAMAM dendrimer yielded a product that contained on
average 96 molecules of Gd-DOTA-4AmP and exhibits an average hydrodynamic volume
consistent with a molecular weight of ~ 140 kDa. The T1 relaxivity of this macromolecular
sensor increases by 122 % (r1 = 10.8 to 24.0 mM−1s−1) over the pH range of 9.5 to 6.576

The Sherry group reported another example of modulating prototropic exchange and water
distribution in the second coordination sphere. The Gd-DOTA tetraamide with pendant
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hydroxypyridyl moieties was used to image changes in pH values (Figure 64B).577

Deprotonation of the amides in those moieties result in intramolecular acid-base pair
interaction with the phenolic protons, leading to a highly organized second hydration sphere.
As a result, the relaxivity of this agent is enhanced at higher pH (pH 8.5) and decreases by
84 % at lower pH (pH 6).

The pH-dependent relaxivity changes of most gadolinium(III) based pH sensors are realized
through modulation of the inner-sphere hydration state, q. An impressive example was
reported by Parker and coworkers who designed a Gd-DO3A derivative with a pendant
sulfonamide (Figure 64C).308 The hydration state changes from q = 0 to q = 2, associated
with the on/off ligation of the sulfonamide nitrogen donor which occurs in a region of great
interest for extracellular pH change. Decreasing the pH from 7.4 to 6.8 resulted in a r1
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enhancement of 35 % when measured in a simulated extracellular anion background and

48 % when measured in human serum. Bis-hydrated Gd-DO3A is known to coordinate
endogenous anions like bicarbonate and phosphate.308, 578–579 The Parker group employed
pendant anionic carboxylate groups (Figure 64C) which served to block anion coordination
and inner-sphere water displacement. The pendant carboxylate groups also resulted in
unexpectedly higher relaxivity than expected based on molecular weight, which is
presumably due to a second sphere effect. In addition, this complex had modest affinity to
HSA while maintaining its pH dependent T1 relaxivity profile.487 Increasing the molecular
weight and thereby the rotational correlation time of the Gd(III) compound through binding
to HSA should dramatically increase the initial r1 enhancement (Gd(III)-complex without
HSA: r1 = 2.4 to 9.0 mM−1s−1 from pH 5 to 8.5, 37 °C, 0.47 T). In fact, binding of the
Gd(III)-complex to HSA resulted in an unexpectedly low r1 enhancement of 42 % (pH 5)
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and 260 % (pH 8.5), respectively, which was attributed to displacement of inner-sphere
water ligands when the complex is bound to HSA. This finding was confirmed using a
derivative with increased HSA affinity.487

Another donor group that can be protonated around neutral pH is phenolate. Sherry and
coworkers showed that a Gd-DO3A derivative with a pendant p-nitrophenolate arm
underwent a switch in hydration state from q = 1 to q = 2 (Figure 64D) as pH was lowered.
580 The protonation and dissociation of the phenolate oxygen donor atom resulted in a 71 %

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enhancement in r1 from pH 9 to 5. Moreover, at low and at high pH, the compound is

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resistant to water ligand displacement by anion coordination.

In a recent study, Giovenzana and coworkers exploited the suitability of Gd-aminoethyl-

DO3A derivatives as a pH-sensitive platform (Figure 64E).581–582 On average, their
compounds showed a percentage enhancement around 100 % in the pH region of 9 to 5, also
provoked by a switch in hydration state.

A major obstacle in applying these systems to image tissue pH is the unknown concentration
of the contrast agents in tissue. In order to measure pH, one must know the relaxivity, and
relaxivity depends on both concentration and T1 change. MRI can measure T1 change but
the challenge of measuring concentration must be addressed.

Gillies, Sherry and coworkers applied a dual injection strategy to determine concentration.
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They administered the pH-independent agent Gd-DOTP and measured the time dependent
signal change. They then administered pH-responsive Gd-DOTA-4AmP (Figure 64A) and
measured the signal change with time.583–584 With the assumption that both agents had
similar tissue biodistribution and pharmacokinetics, the differences in the signal-versus-time
curves for Gd-DOTA-4AmP and Gd-DOTP were attributed to differences in relaxivity. As a
result, the extracellular tissue pH could be successfully mapped in the kidneys in a renal
acidosis model and in a tumor in a brain tumor model.583–584

The same group used a different strategy to map pH in a a rat brain glioma model.585 Instead
of a sequential injection of two agents, this time they mixed Gd-DOTA-4AmP with Dy-
DOTP. Dy-DOTP has extremely low r1 but does exhibit a large R2* effect. By coinjecting an
appropriate mixture, Gd-DOTA-4AmP dominates the ΔR1 effect, while Dy-DOTP
dominates the ΔR2* effect. Concentration of Dy-DOTP was estimated from ΔR2*, and since
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the ratio of Gd:Dy was known, the Gd-DOTA-4AmP concentration could be estimated and
pH determined from the ΔR1 measurement. This again assumed that both contrast agents
had the same distribution and pharmacokinetics. Simply replacing Gd(III) with Dy(III)
would likely give identical pharmacokinetics.

Rather than injecting two different agents, Aime and coworkers proposed a ΔR2/ΔR1
ratiometric method.586 As described above (sections 3.3 & 4.2.b), slow tumbling agents
show increased r1 at low fields but this decreases rapidly with increasing field strengths. On
the other hand r2 is high at low fields and remains constant or increases with increasing field
strength. Thus the r2/r1 ratio increases with increasing field strength but also increases at a
given field strength when τR is increasing. This ratio is independent of concentration. This
group designed a Gd-DOTA functionalized polypeptide (poly-L-ornithine, Figure 65A) that
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exhibits a pH-dependent conformation. At low pH it assembles randomly and exhibits a

shorter τR while at higher pH an ordered α-helix is formed resulting in an increase in τR.
This resulted in a r2/r1 ratio that increased with pH. By measuring ΔR2/ΔR1 (the difference
in relaxation rate in the presence and absence of the agent), the authors could detect pH
changes that were independent of contrast agent concentration. A limitation of this type of
approach is that the r2 relaxivity must be very high to measurably change T2 in tissue where
T2 values are generally very short. That is, although ΔR2/ΔR1 is independent of

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concentration, if the measured ΔR2 and ΔR1 values are small then the error associated with
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the measurement will be very large.

A third strategy to determine concentration independently was reported by Frullano et al.

They incorporated the radionuclide 18F into the framework of the already established pH-
responsive contrast agent Gd-DOTA-4AmP (Figure 65B). By mixing the 18F compound with
the stable 19F analog, they achieved 18F and Gd(III) concentrations suitable for detection by
both PET and MRI. Using a simultaneous MR-PET scanner, they could achieve absolute
quantification with PET while contemporaneously measuring T1 with MRI.587 Combining
the PET (concentration) and MRI (ΔR1) measurements, they could compute relaxivity and
hence obtain pH maps.

Other bimodal activatable pH agents have since been reported. Aime and coworkers
proposed a dual MR-SPECT agent by adding a small amount of the gamma-emitting
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166Ho(III) analog to a previously described pH-responsive Gd-DO3A derivative with a

pendant sulfonamide308 (Figure 65C).588 This system is actually two separate compounds
but it is assumed that the Ho(III) complex would have the same biodistribution and
pharmacokinetics as the Gd(III) analog, which is a reasonable assumption. A limitation of
this approach is that there are no simultaneous MR-SPECT scanners, while MR-PET
scanners are commercially available. For in vivo measurements, the MRI and PET (or
SPECT) measurement must be simultaneous otherwise the concentration measured would
not be reflective of the concentration present when T1 was measured. Another elegant
bimodal MR-PET pH sensor was invented by Tei and coworkers.589 They prepared a
heteroditopic complex that contains Gd(III) as a MRI reporter and 68Ga(III) as the PET
reporter (Figure 65D). The dimeric ligand consists of two different chelating cages: a
DO3A-sulfonamide derivative for the encapsulation of Gd(III) (Figure 65C) and one
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chelator based on AAZTA for the complexation of 68Ga(III). A sulfonamide moiety acts as a
linker between the two chelating cages and provides pH-sensitivity at the same time since
the on/off ligation of the sulfonamide nitrogen donor to the Gd(III) center is pH dependent.
Hence, a switch in hydration state (low pH: q = 2/high pH: q = 0) leads to a percentage
enhancement in r1 of 150 % over the pH range of 8.5 to 5.

6.3.2 pH-responsive CEST contrast agents—CEST agents can also be used as pH-
responsive agents since their mechanism of action is based on the selective saturation of
exchangeable protons, followed by the transfer of the saturated protons to the bulk water
leading to an alteration in water signal intensity. Typically, for a chemical species with labile
protons the exchange rate kex of those protons with bulk water is pH-modulated due to base
catalysis of proton exchange.
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pH-responsive DiaCEST agents: In their seminal 2000 paper describing CEST imaging,
Ward and Balaban also demonstrated that proton chemical exchange agents could be used to
image pH.8 They showed that dihydrouracil (Figure 66A) with its two labile amide protons
can be employed as a pH sensitive imaging agent. They further showed how a ratiometric
method could be used to measure pH in a way that was not dependent on the concentration
of the agent. In general, the magnitude of the CEST effect for one type of exchangeable
proton is different and has a different pH dependence than another exchangeable proton. For

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example in dihydrouracil selective irradiation of each of the amide N-H protons results in a
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different CEST effect. The ratio of these two CEST effects is pH dependent and independent
of concentration. Similar methods could be applied using two different CEST contrast
agents where the signal of one is ratioed against the other. However in this case, the two
agents should have similar pharmacokinetic properties. Longo, Sun, et al. showed that one
does not need two separate exchangeable proton pools for quantitative pH CEST imaging.
They recognized that the efficiency of the saturation power in creating saturation transfer
also has a pH dependence. They showed that by ratioing the CEST effect at different
saturation powers that they could quantify pH using the compound Iobitridol.590

A limitation of diamagnetic CEST agents is their poor sensitivity requiring the

administration of high concentrations to obtain measurable MR contrast. Two strategies can
be adopted to improve their sensitivity with a concentration threshold in the micromolar
range: 1) the chemical shift between the exchangeable proton resonance and the bulk water
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resonance is increased by using strong de-shielding groups like e.g. aromatic rings, halogen
substituents or paramagnetic shift reagents or 2) the number of mobile protons per molecule
is increased by using macromolecular systems like dendrimers and peptides.

Applying the latter strategy, van Zijl and coworkers used a poly-L-lysine scaffold to measure
the pH dependence of its amide proton exchange rates in the physiologic range (Figure
66B).591 The pH dependent NH proton exchange and its relationship to CEST imaging is
illustrated in Figure 65. As shown in the high-resolution 1H-NMR spectrum of poly-L-lysine
(Figure 67, left), at lower pH (pH = 6), the NH proton exchange is rather slow, resulting in a
sharp NH proton resonance. In contrast, at the same pH, the corresponding CEST spectrum
(Figure 67, right) shows only a rather small CEST effect. Increasing the pH to 7.9
accelerates the NH proton exchange, which dramatically broadens the corresponding NH
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proton signal in the 1H-NMR spectrum and in turn amplifies the CEST signal.

X-ray contrast agents are given at very high doses, literally 10’s of grams of these highly
soluble iodinated aromatic compounds. Aime and coworkers recognized that X-ray agents
like Iopamidol, also contain two different amide proton pools with downfield chemical shifts
(Figure 66C), and thus could be used as a pH sensor in MR imaging applications.449
Iopamidol was successfully employed to acquire pH maps of kidney cortex, medulla, and
renal pelvis in healthy mice and to image the pH evolution of an acute kidney injury mouse
model.72, 596 Additionally, in a breast cancer mouse model, tumor pH mapping was
performed using Iopamidol in a combined PET and MR-CEST imaging study.597 Other
approved CT contrast agents with similar structure: Iopromide592 (Figure 66D) and
Iobitridol590 (Figure 66E) were also utilized in vivo for accessing changes in tumor acidosis.
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In another approach, liposomes were filled with L-arginine, a molecule with multiple
exchangeable NH protons (Figure 66F) and subsequently assembled to microcapsules
(LipoCEST). With those pH-nanosensors, transplanted cell viability could be monitored in
vivo.593

Gao and coworkers reported ionizable, tertiary amine-based block copolymers (Figure 66G)
as pH sensors for MR-CEST imaging. Near physiological pH, the polymers form micelles

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turning the CEST signal “off” whereas in an acidic environment the CEST signal is turned
“on” through dissociation of those micelles.594
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Recently, McMahon and coworkers showed that imidazoles (Figure 66H) can also be used as
DiaCEST MRI pH sensors as demonstrated by in vivo mapping of kidney pH in mice.595

pH-responsive ParaCEST agents: One limitation of DiaCEST agents is their small

chemical shift difference from bulk water which can have interference from endogenous
biomolecules that generate CEST in a similar range. A second limitation is sensitivity. For
CEST the chemical shift difference (in Hz) must be greater than the proton exchange rate.
Faster exchange would produce a larger CEST effect but if exchange is faster than the
chemical shift difference then the chemical shifts merge and CEST is impossible. One can
increase the shift difference by moving to higher fields or by turning to ParaCEST agents.
These exhibit a much larger chemical shift difference between the exchangeable proton
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resonance and the bulk water resonance, allowing faster exchange rates to be sampled with
concomitant increased sensitivity.

As outlined in section 3.3, the water exchange rate between the inner sphere of a
paramagnetic center and bulk water is an important parameter that governs CEST sensitivity.
The water exchange rate strongly depends on geometric or steric factors that are created by
macrocyclic ligands encapsulating the paramagnetic ion. Recently, Udugamasooriya and
coworkers reported design principles for Eu(III)-DOTAM-based ParaCEST MR contrast
agents to optimize the water exchange rate and thereby, enhance the CEST sensitivity.598–599

Aime and coworkers investigated the ability of Yb-DOTAM-Gly as a pH-responsive

ParaCEST agent (Figure 68A).600 The compound contains four chemically equivalent amide
protons in close proximity to a paramagnetic ytterbium center. Applying an irradiating
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radiofrequency pulse at the amide N-H resonances at different pH values gave a change in
the net saturation transfer effect from 70 % to 10 % in the pH region of 6 to 8.8. Moreover,
the authors discovered that the concentration of the ParaCEST agent and the saturation
transfer effect are not linearly related. They applied a ratiometric approach by using two
agents differing only in the coordinated lanthanide center: Yb-DOTAM-Gly and Eu-
DOTAM-Gly. In the latter agent, the amide protons are only slightly shifted in a region
where they cannot longer be distinguished from bulk water signals. The CEST spectrum
obtained for a mixture containing both complexes gave three peaks, one for the bulk water,
one for the amide protons associated with Yb-DOTAM-Gly and for the metal coordinated
water protons associated with Eu-DOTAM-Gly. Under the assumption that the saturation
transfer effect of the two different sets of protons (amide protons vs. metal coordinated water
protons) have a different pH dependency, the observed CEST effect depends only on the
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relative concentration ratio of both lanthanide complexes. An improvement of this

ratiometric concept was demonstrated by irradiating two kinds of mobile protons (amide
protons and metal coordinated water protons), that are part of the same paramagnetic
DOTAM-Gly complex. The single-molecule CEST procedure was realized through
employment of lighter Ln(III) ions, like e.g. Pr(III), Nd(III) and Eu(III) where Pr-DOTAM-
Gly (Figure 68A) showed the highest accuracy and sensitivity in its pH dependent behavior
in the pH region 6.0 – 8.0.601–602 However, detecting the CEST effect from metal-bound

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water molecule can require high saturation power that can hamper the safe in vivo
application of those agents because of local heating effects.603
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Pikkemmat and coworkers functionalized different generations of poly(propylene imine)

dendrimers with Yb-DOTAM (Figure 68B) to obtain macromolecular ParaCEST agents,
thereby combining the two strategies (increasing the chemical shift and the number of
exchangeable protons) to improve the sensitivity of the MR contrast agent (vide supra).604 In
this way they could reduce the detection sensitivity by a factor of 15 and observe a 5 %
CEST effect at 20 µM dendrimer solution.

Another paramagnetic DOTAM-Gly derivative: Tm-DOTAM-Gly-Lys (Figure 68C) was

used as pH responsive ParaCEST agent.605 Interestingly, in this study the pH was
determined from the linewidth of the asymmetric magnetization transfer ratio curve, which
has an exponential relation to pH. As a result, pH mapping was successfully realized
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independent from agent concentration and temperature for a given saturation pulse. This
method was also successfully demonstrated in vivo in healthy mice.

Yb-DO3A-oAA (Figure 68D) has two different exchangeable proton pools which also
enables pH imaging via an intramolecular ratiometric approach. This compound was used to
assess pH in a mouse model of MDA-MB-231 mammary carcinoma.603, 607–608

A direct readout of pH by MR imaging is provided by a Eu-DOTAM-Gly derivative (Figure

68E).606 In this elegant example, one of the amide side chains of the DOTAM-Gly ligand is
replaced by a ketone oxygen donor, which in turn is directly conjugated to an ionizable
phenolic moiety. Deprotonation of the phenolic proton leads to conjugation of the resulting
quinone-like structure with the acetyl oxygen donor coordinated to the europium center.
Since the metal-bound water exchange rate is extremely sensitive to alterations in the
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electronic structure of the ligand pendant arms, the CEST signal can be manipulated through
this effect. With a pKa value of 6.7 for the phenolic entity, the agent has ideal properties for
sensing pH in biological systems and shows a large change in exchange frequency over the
pH range of 6.0 to 7.6. Using ratiometric CEST imaging, the pH could be determined
independent of Eu-DOTAM-Gly derivative concentration. Utilization of this frequency-
dependent ParaCEST agent allowed imaging of the pH gradient in kidneys of healthy mice.
609

Aime and coworkers used the Yb(III) analog of the approved contrast agent Gd-HPDO3A as
a pH responsive ParaCEST agent (Figure 3).610 The labile hydroxylic proton shows a pH-
dependent CEST effect in the pH region of 5.0 to 7.0 (37 °C). Moreover, the chemical shift
of the hydroxylic proton is temperature dependent making Yb-HPDO3A a multi-responsive
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ParaCEST agent that can report on pH and temperature simultaneously. Yb-HPDO3A exists
as two major isomeric forms in solution with different chemical shifts for the hydroxylic
protons, and this enables a ratiometric approach in a single-molecule CEST procedure. This
agent was successfully employed for in vivo pH mapping of the tumor region in a melanoma
murine model, as depicted in Figure 69.611

Recently, a pH responsive transition-metal based ParaCEST probe was reported.612 The

high-spin Fe(II) complex of a 2-amino-6-picolyl-appended cyclen ligand shows a pH

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dependent frequency shift at pH 7.7 to 4.8 (37 °C). This shift in the CEST peak correlates
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with the protonation of the unbound pendant 2-amino-6-picolyl units as illustrated in Figure
70. Noteworthy, in this example the CEST peak changes in frequency with changing pH and
not in intensity. In contrast to intensity-responsive contrast agents, frequency-responsive
probes do not require a correction for tissue concentration of the probe.

6.3.3 pH-responsive ParaSHIFT contrast agents—Recently, the Parker group

developed a ParaSHIFT agent for accurate pH and temperature mapping using MR chemical
shift imaging (Figure 71).613 Unlike indirect detection methods, e.g. CEST methodology
where the interaction between the contrast agent and bulk water is detected, in this approach
the MR signal is enhanced by employing paramagnetically shifted resonances and increased
relaxivity from Ln(III) complexes itself. Their Ln-DOTP-like ParaSHIFT agent contains a
pendant pyridine moiety, which in turn is labeled with a homotopic tbutyl group, whose
protons undergo fast relaxation in the field range 1 to 7 T. In direct proximity to the tbutyl
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group, the pyridine ligand also contains a phosphonate group, whose pKa lies in the
physiologically relevant pH region. As a result, the chemical shift of the adjacent tbutyl
group varies as a function of pH. Using the corresponding Dy(III) and Tm(III) complexes in
a ratiometric CEST experiment, the pH and temperature in the liver, kidney and bladder in
healthy mice could be mapped. In comparison to the ParaCEST MR contrast agents
discussed in this section, the sensitivity of this agent is substantially enhanced.
Consequently, the in vivo experiment could be performed using a dose comparable to
clinical MR contrast agents (0.05 mmol kg−1 of a 1:1 mixture of the Dy(III)/Tm(III)
complexes).

6.3.4 pH-switchable contrast agents—Macromolecular and nanoparticle based

agents have been reported that undergo a pH dependent transition resulting in signal change
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and/or accumulation. These agents cannot quantify pH but they may be able to detect
regions of tissue with low pH. For example, Gd(III) containing linear614 and spherical615
copolymers have been developed, that undergo pH dependent conformational changes. At
low pH, the polymers show a higher degree of cross-linking, i.e. the polymers shrink, which
alters the rotational correlation time and hence, enhances r1.

pH sensitive polymeric micelles encapsulating Gd(III) complexes can be disassembled under

acidic conditions.616 The release of the Gd(III) chelates into solution decreases the T1
relaxation time constant of the system. Similarly, acidification of a pH sensitive liposome
provokes the release of Gd(III) chelates into the environment.617

pH responsive manganese oxide nanoparticles have also been reported.618–619 In this

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approach the poor stability of MnO under weak acidic conditions is exploited. The more
acidic tumor microenvironment dissolves MnO, which in turn causes a relaxivity
enhancement by releasing Mn(II) ions into the environment.

In another impressive example, Mn(II) ions were confined in pH sensitive calcium

phosphate nanoparticles comprising a poly(ethylene glycol) shell.620 Upon activation at
lower pH in the solid tumor microenvironment, calcium phosphate disintegrates and Mn(II)-
ions are released into the environment. In a C26 tumor-bearing BALB/C nude mice model, it

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was shown that the released Mn(II)-ions bind to surrounding proteins, which rapidly
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brightens solid tumors and also detects micrometastases in the liver.

Recently, it was shown that SPIOs can be released from hydrogels621 or polymeric
micelles622 under acidic condition, which changes their superparamagnetism and therefore
their T2*-relaxivity.

6.4 Enzyme-responsive molecular imaging agents

The central role of enzymes is to act as biological catalysts, thereby being responsible for
most of the functions in cellular and molecular biology. In many disease processes specific
enzymes are upregulated or become expressed on cell surfaces or in the extracellular space.
Noninvasive sensing of enzymatic activity is, in principle, a powerful tool to detect disease
activity and to monitor the effects of therapeutic interventions. As outlined above, the design
of a contrast agent that is responsive to enzymatic activity benefits from the fact that: 1) the
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catalytic rate of an enzyme can be high enough to make it feasible to modulate the contrast
generating properties of an activatable agent present at micro- to milliomolar concentrations,
and 2) the high specificity of enzymatic reactions so that a change in MR signal can be
attributed to a specific molecular event.

6.4.1 Enzyme-responsive T1/T2*-based contrast agents

Modulation of the hydration state: An established strategy to modulate MR response to
enzymatic activity is to utilize a contrast agent that undergoes a change in hydration state
upon activation.

Meade and coworkers used a Gd(III) chelate to detect oncologically significant β-

glucuronidase.623 Their agent consists of a Gd-DO3A moiety bearing a pendant β-
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glucuronic acid moiety connected by a self-immolative linker to the macrocycle (Figure

72A). The glucuronic acid moiety acts as a mask that can be enzymatically cleaved, which
in turn triggers the “self-destruction” of the linkage into two byproducts, as illustrated in
Figure 72A, resulting in the release of a Gd-DO3A derivative with an altered hydration state.
As a result, the relaxivity in human blood serum decreases by 27 % upon enzyme activation.
Interestingly, performing the same experiment in a buffer mimicking in vivo anion
concentrations leads to an increase in T1 relaxivity by 17 %, which nicely illustrates the
influence of buffer compositions upon the efficacy of an MRI contrast agent.

Lowe and coworkers exploited the known tendency of neutral Gd-DO3A complexes to
avidly bind bicarbonate anions in vivo and form ternary q = 0 complexes.624 They
synthesized a neutral Gd-DO3A derivative with three pendant ethyl ester groups (Figure
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72B). This complex forms a stable, q = 0 complex with bicarbonate but in the presence of an
esterase the ethyl esters are hydrolyzed to liberate three pendant carboxylates. The resultant
anionic complex repels the coordinating anions, which in turn allows water molecules to
bind to the paramagnetic center. The change in hydration state from q = 0 to q = 2 leads to
an increase in T1 relaxivity by 84 % (5.7 to 10.5 mM−1s−1).

Modulation by change in solubility: Insoluble T1 agents have low relaxivity because of

poor access of exchangeable water to the complex. Hoehn and coworkers invented an

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insoluble Gd-DTPA-bis(amide) based, inactive contrast agent (Figure 72C) that can be
internalized into dendritic cells.625 Lipase triggered cleavage of the stearic acid ester groups
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renders the contrast agent soluble with access to bulk water. As a result an increase in MR
signal is observed.

In a similar approach, but in the opposite sense, Lepage and coworkers designed a Gd-
DOTA based agent linked to a PEG-peptide sequence that exhibits excellent water solubility
through the PEG linkage.626–628 A solubility switch is catalyzed through MMP enzymes,
which cleave the PEG peptide, thereby making the chelate insoluble in water. As a
consequence, bulk water access to the chelate is reduced resulting in a decrease in MR
signal. This strategy was applied in MMP-2-rich tumor bearing mice and they showed that
the contrast agent can additionally be accumulated in tumor cells overexpressing the targeted
enzyme.
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Figureueiredo et al. encapsulated Gd-HPDO3A into anionic liposomes. In the presence of

the cationic protein protamine, these Gd(III)-containing liposomes form poorly soluble
aggregates with a relaxivity of only 0.2 mM−1s−1. In the presence of the enzyme trypsin, the
protamine is degraded releasing the liposomes and causing a 9-fold relaxivity increase to 1.8
mM−1s−1.629

Modulation of the rotational correlation time: Another strategy in the design of enzyme
responsive T1 contrast agents is to cause the rotational correlation time to change upon
activation. The general phenomenon of having contrast agent relaxivity increase upon
binding to a receptor was termed RIME (receptor-induced magnetization enhancement)
mechanism. Nivorozhkin et al. synthesized a low relaxivity, pro-RIME agent (Figure 72D),
which consists of Gd-DTPA and a HSA binding moiety that is masked by a HSA shielding
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group. Activation by a human carboxypeptidase B, thrombin-activatable fibrinolysis

inhibitor (TAFI) releases the shielding group resulting in HSA binding, thereby increasing
the relaxivity. Activation in the presence of HSA by micromolar enzyme concentration led to
an increase in r1 of over 150 % at 37 °C.630

In a similar approach, Wang and coworkers developed the enzymatic contrast agent Gd-
DOTA-FPG, which is based on the Gd-DOTA scaffold equipped with a pendant
galactopyranose moiety. This agent can be activated with β-galactosidase. In the presence of
β-galactosidase and HSA, an increase in T1 relaxivity by 60 % was observed.631

Another Gd-DTPA based RIME agent that shows affinity to HSA after activation with the
enzyme ß-galactosidase was invented by Nagano and coworkers. The authors report an r1
enhancement of 60 %.632
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Recently, Wang and coworkers published a Gd(III)-based chelate with a peptide ligand that
is cleaved by legumain, which causes the agent to bind to HSA, boosting the T1 relaxivity of
the agent by 170 %.633

A different strategy to decelerate τR upon activation exploits the enzyme-catalyzed

oligomerization of monomeric contrast agents. Bogdanov et al. synthesized Gd-D-DOTA, a
Gd-DOTA derivative that contains a pendant hydroxyphenol moiety (Figure 73A).635 In the

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presence of peroxidase and hydrogen peroxide, the hydroxyphenol moiety serves as electron
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donor during enzymatic hydrogen peroxide reduction, which in turn generates

hydroxyphenol radicals. The converted Gd(III)-monomers then undergo rapid condensation
into paramagnetic dimers or oligomers. As a result, r1 increases by 200 % (3.75 to 11.50
mM−1s−1, 0.47 T).

A second generation of this probe employs Gd-DTPA units bearing either two
hydroxytryptamide or hydroxyphenetylamide moieties, respectively.636 The latter compound
(Gd-DTPA-diTyr; Figure 73B) was used to sense tyrosinase activity.637 In the presence of
activated tyrosinase, either oligomeric structures were formed (Figure 73B(i)) or in the
presence of tyrosinase and HSA, also paramagnetic complex-protein conjugates can be
formed (Figure 73B(ii)). Both products exhibit an increased rotational correlation time upon
activation. Hence, both products yield an increase in relaxivity.
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Botta and coworkers exploited tyrosine modified Mn(II)-based polyaminocarboxylates as

enzyme-responsive MRI contrast agents (Figure 73C (L1 and L2)).634 In the presence of the
enzyme tyrosinase, r1 increased by 50 % for L1 and by 350 % for L2, respectively. For Mn-
L1, tyrosinase destabilizes the Mn(II) complex which leads to the release of Mn(II) ions and
consequently, to an increase in MR signal. For Mn-L2, it is believed that the enzyme
tyrosinase is catalyzing the formation of oligomeric species, which increases the rotational
correlation time of the agent and hence r1.

The enzyme-catalyzed degradation of a polymer containing MRI contrast agent can be

employed to observe changes in MRI contrast. Degradation of the polymer accelerates the
rotational tumbling time of the MRI agent leading to a decrease in its T1 relaxivity upon
enzyme activation. For example, hyaluronan-Gd-DTPA-beads were attached to aggarose-
avidin beads.638–639 Degradation of the hyaluronan by the enzyme hyaluronidase to lower
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molecular weight fragments led to altered relaxation rates.

Daldrup-Link and coworkers took a different approach in the design of an enzyme-

responsive Gd(III)-based MR contrast agent.640 They designed a caspase-3-sensitive
nanoaggregation MRI probe (C-SNAM) that employs a Gd-DOTA moiety coupled to a
DEVD peptide sequence through an amino luciferin-based linkage that contains a terminal
disulfide entity (Figure 74 left). Enzymatic cleavage of the DEVD peptide and GSH
mediated reduction of the disulfide entity triggers an intramolecular cyclization reaction,
thereby yielding a rigid and hydrophobic product (Figure 74 left), that will subsequently
self-assemble into Gd(III) containing nanoparticles. The prolonged rotational correlation
time of the resulting nanoparticles amplifies the T1 relaxivity by 90 % (r1 = 10.2 mM−1s−1 to
19.0 mM−1s−1 (1 T). Caspase-3 is a common cell apoptosis biomarker.641 To investigate if
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this agent can be applied for in vivo monitoring of transplanted stem cell viability, the
authors transplanted viable FLuc-eGFP-transduced rASCs into an osteochondral defect of
the left distal femur and MMC-treated apoptotic FLuc-eGFP-transduced rASCs into an
osteochondral defect of the right distal femur of athymic female Harlan rats. Intra-articular
injection of C-SNAM provoked significant MR signal enhancement in apoptotic matrix
associated stem cell implants (MASI) compared to viable MASI. The MR signal
enhancement can be attributed to the increased relaxivity of the probe upon activation, but

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also to prolonged tissue retention in apoptotic MASI through its nanoaggregation properties
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(Figure 74 right).

Based on the same approach, the Rao group employed caspase-3-activatable C-SNAMs to
image chemotherapy-induced tumor apoptosis in mice642 and to accurately monitor the
response of tumors to either metronomic chemotherapy or radiation therapy.643

Enzymes that catalyze the aggregation of SPIOs can increase the superparamagnetism of the
nanoparticles and hence, their T2* relaxivity. For instance, the hydrophilic coating of
SPIONs can be cleaved by MMP-2 or MMP-9 enzymes, which leads to aggregation of the
nanoparticles.644–646 Aggregation can be also induced by polymerization of phenolic
SPIONs through the enzyme peroxidase.647 In contrast, also the disaggregation of
nanoparticles can be catalyzed by enzymes, which in turn reduces the superparamagnetism
of the nanoparticles and therefore decrease their T2* relaxivity.648–650
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6.4.2 Enzyme-responsive paramagnetic 19F-based contrast agents—Kikuchi

and coworkers proposed a paramagnetic relaxation-based 19F MRI probe to detect enzyme
activity. Here a Gd(III) complex is placed in proximity to 19F nuclei such that the T2 is
shortened dramatically resulting in attenuation of the 19F MRI signal. If the Gd(III) complex
is released from the vicinity of the 19F nuclei, it will result in an increase in the 19F T2 and
hence enhance the 19F MRI signal. They coupled Gd-DOTA to trifluoromethoxybenzene via
a DEVD peptide linker (Gd-DOTA-DEVD-Tfb) (Figure 75).651 The 19F signal was
broadened into the baseline because of the presence of the nearby Gd(III) ion. In the
presence of the enzyme caspase-3, the DEVD peptide sequence was cleaved releasing the
19F reporter from the agent and resulted in a measurable 19F MR signal. This probe was

further modified to a dual-function agent capable of detecting caspase-3 activity via

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fluorescence measurements and 19F MRI.652

Tethering a 19F reporter molecule to Gd-DOTA via a different enzyme-cleavable linkage

enabled the detection of β-lactamase653 and β-galactosidase654 activity, respectively.

6.4.3 Enzyme-responsive CEST contrast agents

Enzyme-responsive ParaCEST agents: ParaCEST agents can also be utilized for the
detection of enzyme activity. The chemical exchange rate of the labile proton is changed by
enzyme-catalyzed alteration of the covalent bond structure of the ParaCEST agent. As a
result the CEST effect appears, disappears or changes in magnitude.

The first enzyme sensing ParaCEST agent was developed by Pagel and coworkers.655 A Tm-
DOTA derivative was coupled to a DEVD peptide sequence (Figure 76A1), which can be
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selectively hydrolyzed by caspase-3, thereby converting the DOTA-amide into a DOTA-

amine. Under physiological conditions, the disappearance of the CEST effect was observed
upon caspase-3 activation. A limitation of a “turn-off” effect is in quantifying the absence of
signal. In a second study the same group successfully exploited the simultaneous application
of the enzyme-responsive DEVD-Tm-DOTA agent (Figure 76A1) and an unresponsive
ParaCEST agent, Yb-DOTAM-Gly (Figure 68A), to quantify the enzymatic transformation
in a ratiometric approach.656 In this strategy, the unresponsive probe is “always on” and

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accounts for all factors that could alter the CEST effect except for the enzyme to be
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measured. This comparative measurement to improve the detection of enzyme activity is

referred to as “catalyCEST MRI”.

The Pagel group extended this method for the in vivo detection of urokinase plasminogen
activator (uPa) in a Capan-2 pancreatic tumor mice model where Cbz-GGR-Tm-DOTA
(Figure 76A2) served as a uPa responsive probe and Eu-DOTAM-Gly as control probe.657
This proof-of-concept study represents the first report of in vivo catalyCEST MRI.
Replacement of the peptide sequence of a Tm-DOTA derivative enabled the detection of
cathepsin-D activity.658

Additionally, modularly designed Yb-DOTA derivatives have been used as enzyme sensing
ParaCEST agents. For instance the Yb-DOTA complex in Figure 76B is coupled to a β-
galactose ligand via a self-immolative linker. Selective cleavage of the sugar by β-
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galactosidase results in elimination of the linker group and the amide bond is hydrolyzed to
an amine thereby generating a CEST effect.659

Yb-DO3A-oAA-TML-ester (Figure 76C) is capable of sensing esterases, which cleave the

ester of the chelator (shown in blue, Figure 76C).660 The tri-methyl lock moiety can then
undergo lactonization (shown in green, Figure 76C) also resulting in the conversion from an
amide into an amine in the Yb-DO3A-oAA contrast agent. A similar probe Yb-DO3A-oAA-
TML-Q is activated by DT-diaphorase.661

It was also shown that for enzyme activated ParaCEST agents, the CEST effect can be
generated through the opposite reaction, i.e. formation of an amide bond from an amine
group. Conjugation of Tm-DO3A-cadaverine (Figure 76D) to albumin by transglutaminase
produced a “turn-on” CEST effect.662 Concurrently, a reduced CEST signal from albumin
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could be observed.

Enzyme-responsive DiaCEST agents: DiaCEST agents have also been developed to detect
enzyme activity. Gilad and coworkers reported that the enzyme cytosine deaminase (CDase)
catalyzed deamination of cytosine to uracil or F5C to 5FU, respectively (Figure 77A).663 As
a result, the previously observed CEST effect is “switched off”.

Another example is the DiaCEST peptide (LRRASLG)8 which shows high CEST contrast
due to its basic, positively charged lysine and arginine residues. Phosphorylation of this
peptide through the enzyme protein kinase A creates a strong negatively charged group
within the molecule, which slows down the water exchange between the exchangeable
protons and the bulk water protons. Consequently, the authors observed a reduced CEST
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effect in in vitro experiments.664

Recently, (Phe-Arg)-4-amino-2-hydroxybenzoic acid (Figure 77B) was proposed as a

catalyCEST MRI agent that contains an enzyme-responsive entity and also a non-responsive
reference. After cleavage of the dipeptidyl ligand through the enzyme cathepsin B (shown in
blue, Figure 77B), the aryl-amide proton is converted into an aryl-amine proton, i.e. the
CEST signal observed for the aryl amide proton disappears. The salicylic acid moiety is
largely unresponsive to cathepsin B activity and acts as non-responsive reference.665

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The modular design of salicylic acid derivatives (Figure 77C) facilitated several “turn-on”
catalyCEST MRI agents capable of sensing sulfatase666, alkaline phosphatase667, and the
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presence of the two enzymes esterase and sulfatase at the same time.668 Additionally, a
glutamyl derivative of salicylic acid (Figure 77D) was successfully employed in mouse
models of human ovarian cancers to selectively detect γ-glutamyl transferase.669

6.5 Redox potential-responsive molecular imaging agents

The intra- and extracellular redox environment is tightly regulated in order to maintain
normal physiological processes. In disease states the redox environment can become
disrupted, so that altered redox is generally associated with wide range of
pathophysiological conditions. The redox environment is regulated by metabolites, including
NAD/NADH, peroxides, thiol/disulfides (e.g. glutathione), nitric oxide, and oxidase
enzymes. Activatable MR contrast agents can be designed to be altered by redox active
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metabolites. The design of redox sensitive probes can involve a change in oxidation state of
the ligand or the metal center to provide the desired change in MR contrast with altered
redox environment.

6.5.1 Redox potential-responsive T1 contrast agents—One strategy to sense the

redox changes is the modulation of the hydration state of the contrast agent upon activation.
Louie and coworkers employed this strategy by introducing a merocyanine moiety into a
Gd-DO3A complex in a way that the oxygen donor of merocyanine could coordinate to the
Gd(III) center (Figure 78A).670–671 After treatment with NADH, the merocyanine group was
converted into its spirooxazine isomer, incapable of coordinating the metal center and hence,
leaving space for a water molecule to coordinate to the Gd(III) ion. As a result the hydration
state of the complex changed from q = 1 to q = 2, thereby increasing r1 by 55 %.
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Iwaki et al. showed that Gd-4NO22MeOSA (Figure 78B) could undergo reduction to the
corresponding aminobenzenesulfonamide in the presence of rat liver microsomes under
hypoxic conditions, but not under normoxic conditions.672 The agent consists of a Gd-
DO3A derivative with a pendant nitrobenzenesulfonamide where the sulfonamide nitrogen is
deprotonated and coordinated to the Gd(III) center at neutral pH. Reduction to the
aminobenzenesulfonamide raises the pka value of the sulfonamide nitrogen resulting in
protonation and dissociation from the Gd(III) ion. As a result, the hydration state of the
complex switches from q = 0 to q = 2 and r1 increases by 70 %.

Goldsmith and coworkers developed Mn(II)-H2qtp1 (Figure 78C), a Mn(II) based complex
with a redox-active ligand.673–675 The hexadentate ligand contains a dihydroxybenzyl group
that can be oxidized to the weaker coordinating benzoquinone using hydrogen peroxide. As
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a consequence, r1 increases from 4.73 to 5.3 mM−1s−1, presumably because of greater

aquation of the Mn(II) ion. By adding a second dihydroxybenzyl group into the ligand
scaffold, r1 could be further increased from 5.46 to 7.17 mM−1s−1.

Another example for a redox potential-responsive agent is Gd-DO3AS-Act (Figure 78D).676

The Gd-DO3A based agent bears a 2-pyridyldithio functionality readily available for
disulfide exchange reactions. An exchange reaction with glutathione introduces an additional
pendant carboxylic acid moiety within the molecular scaffold, which can coordinate the

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Gd(III) center. This in turn leads to the displacement of the inner-sphere water molecule and
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the hydration state of the complex switches from q = 1 to q = 0, thereby decreasing r1 by

50 %.

Another thiol-bearing Gd-DOTA-monoamide derivative (Gd-LC6-SH) (Figure 78E) was

attached to HSA via a disulfide bridge.677–679 When bound to albumin the T1 relaxivity of
the gadolinium(III) complex is higher than in the unbound form due to the slower tumbling
rate of the Gd(III)-HSA adduct. In a reducing environment, the gadolinium(III) complex is
cleaved from HSA by reduction of the disulfide bond, which decreases r1 by 55 %. In a
proof-of principle study, Gd-LC6-SH was applied as a MR reporter of tumor redox status in
in vivo experiments with mice bearing Mia-PaCa-2 or NCI-N87 tumor xenografts.680

Conjugation of thiol-bearing Gd-DO3A derivatives to the surface of liposomes via disulfide

linkage led to paramagnetic, supramolecular adducts with slow rotational correlation times.
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The disulfide linkage makes them sensitive to reducing environments, which was shown in
in vitro experiments. Free thiol mediated cleavage of the Gd(III) complexes from the surface
of the liposomes accelerated τR and decreased the r1 from 13.6 to 6.5 mM−1s−1.681

Aime and coworkers first proposed the utilization of the Mn(II)/Mn(III) couple as an
activation mechanism for the development of redox sensitive T1 contrast agents.382 They
used the well known Mn(III)-TPPS (Figure 78F) complex which had been used as a contrast
agent for tumor imaging.682–685 They also prepared the Mn(II) complex by reducing with
dithionite. The relaxation mechanism is different for the two complexes at 0.47 T: the
dominant correlation time for the Mn(III) complex is the electronic relaxation time, while
for Mn(II) it is the rotational correlation time. At 0.47 T, Mn(III)-TPPS has a higher
relaxivity than Mn(II)-TPPS. However when poly-β-cyclodextrin is added, host-guest
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complexes form resulting in a longer rotational correlation time. This has a dramatic effect
on the relaxivity of Mn(II)-TPPS (r1 = 40.8 mM−1s−1) but much less so with Mn(III)-TPPS
(15.2 mM−1s−1). In the presence of 40 torr pO2 (pO2 of venous blood vessels is approx. 40
torr) the Mn(II)-TPPS:CD adduct was completely oxidized.

Loving et al. took a different approach to the Mn(II)/Mn(III) couple.686 They showed that
the HBET ligand could stabilize both oxidation states, but the Mn(II)-HBET complex
(Figure 78G) is seven coordinate, q = 1, while Mn(III)-HBET is six coordinate q = 0. In
addition to the change in hydration state, the difference in spin state will also result in a
lower relaxivity for the Mn(III) form. At pH 7.4, 37 °C, 1.4 T, r1 was 2.8 mM−1s−1 in the
divalent state and 1.1 mM−1s−1 in the trivalent state. Loving et al. further showed that this
system could be reversibly oxidized with hydrogen peroxide and concomitant signal loss or
reduced with glutathione and concomitant signal increase. This work was further extended
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by synthesizing a series of derivatives where the electronic substituent on the aromatic ring
was varied from electron withdrawing to electron donating.371 They also examined the effect
of changing from an ethylenediamine to a cyclohexamine diamine backbone on relaxivity,
thermodynamic stability, pH-dependent complex speciation, hydration state, water exchange
kinetics of the Mn(II) complexes, and pseudo-first order reduction kinetics. They found that
they could improve the relaxivity turn-on to a factor of 7.5 (r1 = 0.5 mM−1s−1 for Mn(III)-
CyHBET compared to 3.3 mM−1s−1 for Mn(II)-CyHBET).

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Mn-HBET and Mn-CyHBET provided strong proof of concept for Mn complexes as redox-
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activated MRI contrast agents but the Mn(II) oxidation state is favored exclusively in blood
plasma. Mn(II) and Mn(III) favor disparate and distinct coordination environments and
developing a ligand capable of supporting complexes where both oxidation states can persist
in blood plasma is challenging. Mn(II) is best stabilized by poly-amino/carboxylate/pyridyl
ligands like EDTA or N,N’-bis(pyridyl)-ethylenediaminediacetic acid (BPED), whereas
Mn(III) favors more electron releasing ligands phenolate containing ligands like HBED,
Figure 43. For example, Mn(III)-EDTA will decompose via disproportionation to Mn(II) and
Mn(IV),687 whereas the Mn(II) complex of HBED will spontaneously oxidize to Mn(III)
upon O2 exposure.688 Mn-HBET was designed as an initial compromise between the Mn(II)
and Mn(III) ligand preferences but the redox potential of Mn-HBET is >500 mV greater
than cysteine/cysteine disulfide redox couple. Attempts to depress the Mn(II)/Mn(IIII) redox
potential via incorporating electron releasing substituents onto the phenolato-O donor
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resulted in ligand-metal auto-redox upon oxidation to Mn(III). One solution was to develop
a chelator that was capable of isomerizing between binding modes that favor Mn(III)
(BPED) or Mn(II) (HBED) exclusively. The chelator JED (Janus HBED/BPED, Figure 78H)
was demonstrated to support stable and high-relaxivity complexes of both Mn(III) and
Mn(II) in blood plasma. Switching between the Mn(III) and Mn(II) oxidation states resulted
in a 9-fold increase in blood plasma r1 at 1.4T, 37 °C. Importantly, a large Mn(II) vs. Mn(III)
r1 differential was maintained up to 11.7T.689 Interconversion between the oxidation states
can be triggered in the presence of peroxidase enzymes or addition of excess L-cysteine.

A novel procedure for the MR based assessment of hypoxia was reported by Aime and
coworkers.690 In this innovative example, the combined utilization of Gd-DOTP- and Gd-
HPDO3A-labeled red blood cells made it feasible to map tumor hypoxia in a transplanted
breast tumor mouse model. Gd-DOTP-labeled red blood cells act as a vascular oxygenation-
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responsive agent. Gd-DOTP shows 5 times higher affinity for deoxy-hemoglobin in

comparison to oxy-hemoglobin. As a result, Gd-DOTP-labeled red blood cells show far
higher T1 relaxivity in hypoxic regions than in normoxic areas. Gd-HPDO3A-labeled red
blood cells were used to furnish the local concentration of red blood cells. In the
corresponding in vivo study, firstly Gd-HPDO3A-labeled red blood cells were administered
and MRI vascular volume maps were acquired. Afterwards, Gd-DOTP-labeled red blood
cells were injected into the same animal and T1-weighted images were acquired. The ratio
between those two images gave a relative MRI deoxygenation map that reports on O2
content independently from vascular volume (Figure 79).

6.5.2 Redox potential-responsive ParaCEST contrast agents—Pagel and

coworkers proposed Yb-DO3A-oAA (Figure 80) as a responsive ParaCEST agent capable of
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detecting NO in the presence of O2.691 This compound exhibits two CEST effects, caused
by the exchangeable protons from the amine and the amide hydrogens, respectively. In the
presence of NO and O2, the compound undergoes an irreversible covalent conversion to a
dinuclear species bridged through a triazene (Figure 80A), thereby losing both CEST effects.

Ln-DOTA-tetraamide derivatives exhibit sufficiently slow water exchange kinetics that the
shifted, coordinated water ligand can act as a CEST reporter. Water exchange kinetics can be
tuned by altering the substituents on the amide groups either with respect to charge,

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lipophilicity, or electronic structure.451 Sherry and coworkers developed a series of Eu(III)-

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based ParaCEST agents where oxidation or reduction altered the water exchange rate and, in
turn, the CEST signal. These authors reported an activatable agent with two pendant N-
methylquinolinium moieties,692 designed to mimic the NADH/NAD+ couple. In its oxidized
form (Figure 80B (blue)), the agent is nearly CEST MRI silent but was “turned-on” after
irreversible reduction with NADH to the corresponding dihydroquinoline derivative (Figure
80B (red)). The larger CEST signal upon reduction is attributed to the slower water
exchange rate of the reduced species.

Another agent utilized a pendant 9-anthryl group (Figure 80C (blue)) that acts as a specific
reactive center for singlet oxygen (1O2), a highly unstable reactive oxygen species (ROS)
that is associated with cancer and cardiovascular diseases.201 Oxidation of the 9-anthryl
moiety with 1O2 irreversibly forms the stable endoperoxide derivative (Figure 80C (red)).
Like in the case of Yb-DO3A-oAA (vide supra), the non-equilibrium probe design was
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chosen to form a sufficient amount of oxidized species to allow CEST detection considering
the short-live and low concentration of 1O2. Binding of singlet oxygen successfully shifted
the position of the metal-bound water CEST peak. Moreover, ratiometric CEST imaging in
in vitro systems revealed the potential of this agent to sense 1O2 with high chemical
specificity, rapid reaction kinetics as well as high kinetic and thermodynamic stability.

A different mechanistic approach by the Sherry group was to employ two redox-sensitive
nitroxide free radicals, as amide substituents (Figure 80D). The paramagnetic nitroxide
radicals serve to shorten the T1 relaxation time of bulk water protons, and this short T1 has
the effect of nullifying the CEST effect because water relaxes too quickly upon saturation.
184 Nitroxides can be rapidly oxidized or reduced to their diamagnetic derivatives, e.g. under

hypoxic conditions reduction of nitroxide radicals has been demonstrated. Formation of the
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diamagnetic nitroxide (Figure 80D, (red)) through either a biological or chemical means
would lengthen the T1 of bulk water protons and restore the CEST signal. Injection of the
probe in healthy mice showed that the probe was still intact after excretion in the bladder,
i.e. the nitroxide radicals were not reduced by biological processes. Subsequent
administration of the reducing agent L-ascorbic acid activated the agent as evidenced by the
strong CEST signal in the bladder.

LipoCEST agents contain a fast exchanging, paramagnetic Ln(III) shift reagent encapsulated
inside a liposome. If the rate of water exchange across the liposomal membrane is slow
enough, then there will be two water resonances observed in the NMR spectrum: the bulk
water outside the liposome and the shifted water inside the liposome. Selective saturation of
the intraliposomal resonance generates CEST with high sensitivity due to the large number
of water molecules inside the liposome.293, 694 Terreno and coworkers developed a disulfide-
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based, redox sensitive lipoCEST agent.695 They attached a Gd-DO3A derivative to the outer
surface of the liposome via a disulfide linker (Figure 81). The Gd(III)-modified liposome is
CEST silent because of the T1-shortening effect of the Gd(III), but redox-mediated disulfide
reduction results in release of the Gd(III) complexes and restoration of the lipoCEST signal
(Figure 81). It is worth noting that the linkage between the Gd(III) complex and the
lipoCEST agent is easily exchangeable, so that these agents could be made sensitive to a
variety of other stimuli through sophisticated choice of the biodegradable linker.

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Allen and coworkers demonstrated that the Eu(II)/Eu(III) redox switch can be exploited for
molecular imaging.405 The authors encapsulated a Eu(II)-cryptate (Eu(2.2.2)2+) in
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liposomes to form a dual mode contrast agent, which shows T1 enhancement in the reduced
form and also exhibits a CEST effect. Oxidation of divalent Eu(2.2.2)2+ to Eu(2.2.2)3+
silenced the T1 enhancement but maintained the CEST effect with similar intensity.
Interestingly, the CEST effect was independent from the europium concentration within the
liposome, suggesting that the CEST effect is due to the liposome membrane itself rather than
the europium compounds within. Detection of both the T1 enhancement and CEST effect
implies that the agent has not responded to an oxidative trigger, but loss of T1 enhancement
indicates irreversible activation by oxidation. The liposomes were kinetically stable for both
oxidation states of europium.

Morrow and coworkers reported a triazamacrocyclic cobalt complex with exchangeable

protons located on pendant pyrazole groups (Figure 80E).693 The paramagnetic Co(II) center
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causes a large shift of these exchangeable protons relative to the water resonance, whereas
the in the oxidized form with diamagnetic Co(III), these resonances are much closer in
frequency to bulk water. The system exhibits a reversible Co(II)/Co(III) redox couple that is
tunable over the biologically relevant range of −80 to −280 mV (versus normal hydrogen
electrode) by varying the ligand substituents.

6.6 Metal ion-responsive molecular imaging agents

Metal ions such as Ca(II), Cu(II), and Zn(II) play an important role in cell signaling. The
ability to noninvasively image ion flux would enable studies to elucidate organ function and
also to identify pathological states characterized by altered metal ion homeostasis. There are
a number of challenges in developing metal ion-responsive contrast agents. In addition to the
challenges of sensitivity and appropriate dynamic range that affect all responsive agents, for
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detecting metal ion flux one requires specificity for the ion of interest and, perhaps more
importantly, sufficiently fast reversible binding kinetics to sense metal ion concentration
change in real time. For T1 agents, the strategies of coupling metal binding to modulation of
the rotational correlation time or to modulation of the hydration number have been
employed. There have also been ion-responsive ParaCEST agents and SPIONs reported in
recent years.

6.6.1 Zn(II)- responsive MR contrast agents—The first Zn(II) responsive contrast

agents were published by Nagano and coworkers.696–697 They synthesized a series of Gd-
DTPA-bisamide complexes sensitive to Zn(II) ions and their best candidate is depicted in
Figure 82A. In the presence of Zn(II) ions, the authors posit that the chelator undergoes a
geometrical reconfiguration to coordinate Zn(II), which in turn displaces the inner-sphere
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water molecule. As a consequence, r1 decreases from 4.8 to 3.4 mM−1s−1 after addition of 1
equivalent of Zn(II). The binding affinity for Zn(II) was not determined in this study, but
notably, this sensor selectively responds to Zn(II) ions over Na(I), K(I), Ca(II) and Mg(II).

Meade and coworkers reported a Gd-DO3A derivative with a pendant iminodiacetate group
for zinc binding (Figure 82B).698–699 Coordination of Zn(II) changes the hydration number
for Gd(III) from q = 0 to q = 1, i.e. the sensor “turns on” in the presence of zinc with a fairly

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high r1 change of 121 %. This sensor was selective for Zn(II) over Na(I), K(I), Ca(II) and
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Mg(II), but also showed some response for Cu(II). In general, it is believed that a
simultaneous Cu(II) response does not interfere in in vivo experiments since the Cu(II)
concentration is typically much lower in tissue than the Zn(II) concentration. The Kd for
Zn(II) is 240 µM and in vitro MR images showed that zinc concentrations as low as 100 µM
could be detected with this agent, which is within the physiologically relevant region. The
Zn(II) concentration in vesicles of certain types of glutamatergic neuronal, prostate, and
pancreatic cells can reach 300 µM.571 However, the detection of lower zinc levels in vivo is
still desirable.

Esqueda et al. reported the Gd-DOTA-bisamide agent Gd-CP027 that binds up to two
equivalents of Zn(II) using pendant N,N-bis-(2-pyridyl-methyl) ethylene diamine (BPEN)
groups as amide substituents (Figure 83A).700 In the presence of Zn(II) in a buffer system,
Gd-CP027 displays only a modest 20% increase in r1. However, in the presence of Zn(II)
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and HSA, a large 165 % r1 change is observed (r1 = 6.6 to 17.4 mM−1s−1). Coordination of
Zn(II) to Gd-CP027 enables its binding to a subdomain of HSA, which increases the
rotational correlation time of the agent and hence its relaxivity (Figure 83B). In the absence
of Zn(II), Gd-CP027 shows no affinity for HSA. This sensor exhibits sensitivity for Zn(II)
ions over Ca(II) and Mg(II), but does respond to Cu(II). The authors demonstrated in in vitro
experiments, that in the presence of HSA, zinc concentrations as low as 30 µM could be
detected and a remarkably high binding affinity for zinc (Kd = 33.6 nM) was reported.
Ultimately, Gd-CP027 was applied in vivo. The authors were able to image the glucose
stimulated Zn(II) secretion in the mouse pancreas or prostate gland.701–702 The zinc content
in prostate cancer tissue is approximately sixfold decreased in comparison to healthy
prostate tissue. Using Gd-CP027, it was feasible to distinguish between healthy and
malignant mouse prostate by imaging the differential secretion of Zn(II). As illustrated in
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Figure 83C, treatment of healthy mice with glucose and Gd-CP027 led to clear contrast
enhancement in the healthy mice prostate tissue (bottom). On the other hand, stimulation of
prostate tumor-bearing mice with glucose revealed that different stages of tumor
development could be discriminated from each other (middle & top) since the zinc
concentration decreases with progressing disease.

ParaCEST agents can also be used as Zn(II)-sensitive MR contrast agents. For instance, a
Eu-DOTAM derivative equipped with two BPEN units for Zn(II) binding was reported by
Sherry and coworkers (Figure 84A).703 In the absence of Zn(II) ions, the agent shows an
intermediate to slow water exchange between the Eu-OH2 and bulk water molecules.
Coordination of Zn(II) ions through the four pendant pyridine units leads to a mononuclear
complex close to the europium center. Unexpectedly, the water exchange at the europium
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center accelerated resulting in disappearance of the CEST signal. The effect was even more
dramatic at higher pH (pH 8) suggesting that there is a water ligand also coordinated to
Zn(II) that can be partially deprotonated at pH 8 to give Zn-OH. The close proximity of the
Zn-OH species to the Eu(III)-bound water molecule is believed to catalyze the prototropic
exchange between the Eu(III)-bound water molecule and bulk water (Figure 84B). CEST
imaging experiments revealed that the agent is selective for Zn(II) over Ca(II) and Mg(II)
ions and the effective sensitivity range is estimated to be 5 to 120 nM.

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Water-soluble Mn(III)-porphyrins were also employed as MRI based zinc sensors (Figure
84C).704 Notably, the porphyrin TPPS-BIPEN, Figure 84C is a fluorescent sensor for Zn(II).
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When Mn(III) is coordinated by the porphyrin ligand and the complex binds Zn(II), the
contrast agent is able to permeate cell membranes making it the first cell permeable MRI
sensor for metal ions reported. Due to an unknown mechanism, the T1 relaxivity decreases
from 8.70 to 6.65 mM−1s−1 (24 %) upon addition of 1 equivalent of Zn(II). Interestingly, this
trend reverses when the Mn(III)-based agent is treated with Zn(II) ions in a cellular context.
Incubation of HEK-293 cells with Mn(III)-porphyrin led to an increase in the T1 relaxivity
in the presence of Zn(II) ions. Additionally, T2-weighted images of these cells showed also
an increase in relaxivity associated with zinc addition. In a follow-up study, the authors
reported Zn(II) sensitivity of their contrast agent when it was directly injected into the brain
of rats.705

6.6.2 Ca(II)- responsive MR contrast agents—Meade and coworkers reported the

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first Ca(II) ion sensitive contrast agent Gd-DOPTA (Figure 85A).706–707 Gd-DOPTA
consists of two Gd-DO3A units bridged by a modified 1,2-bis(o-aminophenoxy)ethane-
N,N,N’,N’-tetraacetic acid (BAPTA) unit, which is known to have specificity for Ca(II) over
other metals. In the absence of Ca(II), the anionic carboxylate oxygen donor atoms of the
BAPTA unit bind to the gadolinium(III) centers yielding a q = 0 complex with r1 = 3.26 mM
−1s−1. In the presence of Ca(II), the BAPTA carboxylate groups preferentially coordinate

Ca(II), thereby allowing water molecules to coordinate the gadolinium(III) centers, which
changes the hydration state per Gd(III) ion from q = 0 to q = 1. As a result r1 increases by
80 % to 5.76 mM−1s−1. Gd-DOPTA shows selectivity for Ca(II) over Mg(II) and a binding
affinity for calcium of Kd = 0.96 µM. In the human body, the intracellular Ca(II)
concentration is in the micromolar range while the extracellular Ca(II) concentration is in
the millimolar range. Thus, Gd-DOPTA has a MRI response in the intracellular
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concentration range and a possible application of such an agent is to sense Ca(II)

fluctuations in biological settings. This probe was further developed by masking the BAPTA
units with ethyl esters to firstly increase cell labeling and prevent extracellular Ca(II)
binding (Figure 85B).708 Once inside the cell, the ethyl ester groups can be cleaved by the
enzyme esterase, thereby unmasking four carboxylates that coordinate to the Gd(III) centers
and block access to coordinated water ligands. Subsequent intracellular Ca(II) binding
results in an increase in hydration number and facilitates an increase in relaxivity by 66 %.
This probe design allows for discrimination between extra- and intracellular Ca(II) ions.

Gd-DOPTRA, a Gd(III)-based Ca(II) sensor with the highest “turn-on” response based on a
switch in hydration state was reported by Dhingra et al. (Figure 86A).709 The agent consists
of a Gd-DO3A unit equipped with a pendant calcium-responsive o-aminophenol-N,N,O-
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triacetate (APTRA) unit. In the absence of Ca(II)-ions, the APTRA unit can coordinate to
the Gd(III) center yielding a q = 0 complex. In the presence of Ca(II), the APTRA unit
preferably coordinates Ca(II) ions yielding a q = 1 complex resulting in a doubling of r1 (3.5
to 6.9 mM−1s−1). Gd-DOPTRA shows selectivity for Ca(II) over Mg(II) and Zn(II) and
exhibits a binding affinity for calcium in the micromolar range (Kd = 11 µM). However in
biological media at 37 °C, r1 only increased by 25 % in the presence of Ca(II). Presumably,

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this attenuated response is due to anion binding to the Gd(III) center in the presence of
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Ca(II) that also blocks water access and dampens r1.

Kubíček et al. described Gd-DO3APBP which is a Gd-DO3A derivative with a pendant

biphosphate ligand as a Ca(II) sensor (Figure 86B).710 Ca(II) binding to the bisphosphonate
creates a coordination oligomer that has a slower tumbling time than the corresponding
monomer and hence, a higher T1 relaxivity. However, Gd-DO3APBP is not selective for
Ca(II) over Mg(II) or Zn(II). In the presence of ≥3 equivalents of either of these ions, r1
increases by 200 to 500 %.

An exemplary ParaCEST agent able to sense Ca(II) is based on a Yb-DOTA-tetraamide

chelator with four iminodiacetate arms (Figure 86C).711 In the absence of Ca(II), a CEST
effect is observed for this compound originating from the slow exchange of the amide
protons as well as the Yb-OH2 water ligand. Upon coordination of Ca(II) ions via the
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pendant iminodiacetate groups, the exchange of the amide protons is slowed down resulting
in a marked reduction in the CEST effect. A similar effect is observed in the presence of
Mg(II).

Another strategy for sensing Ca(II) ions was proposed by Jasanoff and coworkers.712–713
These authors developed calmodulin modified SPIONs for calcium detection using changes
in T2 relaxation. When the calmodulin groups bind Ca(II) this results in an aggregation of
the nanoparticles. Particle aggregation is a well known phenomenon that results in increased
r2. Their best candidate exhibits a r2 change from 200 to 34 mM−1s−1 which is in a
convenient range for intracellular calcium detection (EC50 = 1.4 µM). However, the cell
membrane impermeability of this agent greatly limits its in vivo application.

Angelovski and coworkers designed a system that combined a change in hydration number
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with a change in rotational correlation time to amplify Ca(II) sensing.714 They incorporated
an amphiphilic calcium sensor into a liposome bilayer. The sensor consists of a Gd-DO3A
chelate coupled to a Ca(II) chelator, which is in turn coupled to a lipophilic alkyl chain
(Figure 87). The paramagnetic liposome system exhibits r1 = 7.3 mM−1s−1 (15 °C, 20 MHz)
in the absence of Ca(II) ions at physiological pH, but after saturation with Ca(II) ions, r1
increases by a remarkable 400 % (r1 = 38.1 mM−1s−1). This can be explained through
additive effects that occur upon Ca(II) binding. Coordination of Ca(II) through the
incorporated calcium chelator causes a conformational change within the amphiphilic ligand
that on the one hand changes the hydration state of the Gd-DO3A unit from q = 0 to q = 1.
At the same time, the fast intramolecular rotation is slowed down, which further enhances r1
(Figure 87). To the best of our knowledge, this has been the highest change in T1 relaxivity
for a Ca(II)-responsive Gd(III)-based contrast agent at physiological pH reported so far.
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6.6.3 Cu(I)/Cu(II)- responsive MR contrast agents—Chang and coworkers

developed a variety of MRI-based copper(I) sensors.715–716 The authors utilized Gd-DO3A
derivatives coupled to acetate or thioether-rich receptor ligands that are capable of
coordinating copper ions. In this molecular framework, the Gd(III) center is shielded from
coordinating water molecules in the absence of copper ions. Here a picolyl group
coordinates Gd(III) in the absence of Cu(I), but is displaced by copper binding resulting in

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coordination of a water ligand to Gd(III), i.e. the hydration state of the complex changes
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from q = 0 to q = 1 or 2 (Figure 88A). For the thioether-tethered Gd-DO3A chelate, that is

depicted in Figure 88B, the highest enhancement in r1 exploiting a hydration state
mechanism has been observed after treatment with 1 equivalent of Cu(I). The T1 relaxivity
increases by 360 % from 1.5 to 6.9 mM−1s−1. Additionally, this sensor shows impressive
selectivity for Cu(I) over Na(I), K(I), Ca(II), Mg(II), Fe(II), Fe(III), Cu(II), and Zn(II) and
remarkably high affinity for Cu(I) (Kd = 0.26 pM). However, the presence of coordinating
anions can compete with the inner-sphere water molecule and affect the relaxivity response
to Cu(I) binding. The authors overcame this problem by installation of additional
carboxylate groups on the periphery of the molecular scaffold (Figure 88C), a known
strategy to reduce the sensitivity to biologically abundant coordinating anions.717 By
maintaining the selectivity and the sensitivity for Cu(I), the relaxivity response to Cu(I) ions
was only slightly affected by this modification. In the absence of Cu(I) ions, the contrast
agent exhibits a relatively low T1 relaxivity (2.6 mM−1s−1), whereas addition of Cu(I)
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triggers a 340 % enhancement in r1 to 11.4 mM−1s−1. As a second modification, the authors

conjugated their Cu(I) sensor to an octaarginine peptide (Figure 88D) to enable cellular
uptake of the contrast agent.718 The agent shows a Cu(I) induced r1 enhancement of 220 %
(3.9 to 12.5 mM−1s−1). In vitro experiments demonstrated that this Cu(I) sensor can report
on labile copper pools in a disease model where differences in copper accumulation between
cells bearing a mutant copper transporter and wildtype cells could be detected.

Chen and coworkers developed Gd-QDOTAMA as a Cu(II) responsive agent that consists of
a Gd-DO3A derivative with a pendant quinoline based ligand, chosen to selectively
coordinate Cu(II) ions (Figure 88E).719 Cu(II) coordination provokes a switch in the
hydration state of the agent from q = 1 to q = 2. Upon addition of 1 equiv. of Cu(II) ions, r1
increases from 4.27 to 7.29 mM−1s−1 (71 %). This agent shows high selectivity for Cu(II)
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over Na(I), K(I), Ca(II), Mg(II), Fe(II), Fe(III), and Zn(II). The authors estimate the Cu(II)
dissociation constant to be Kd = 0.16 nM.

6.7 Other responsive molecular imaging agents

6.7.1 Neurotransmitter-responsive MRI contrast agents—Neuroscience, the
study of the brain and nervous system, is inspired by the desire to understand the complexity
of the brain and behavior. To facilitate its various functions, the brain contains billions of
neurons which interact to form circuits and giving rise of a complex network. The major
mode for communication between these nerve cells is mediated through chemical
neurotransmission.720–721 Consequently, neuroimaging techniques can provide
indispensable insights into the mechanisms of neural functions both in health and disease.
722–724 Such studies may enhance the understanding of physiology and pathophysiology of
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the brain and as a consequence improve the diagnosis and treatment of brain disorders.

Recently, Jasanoff and co-workers developed the first neurotransmitter sensitive MRI
contrast agents which are based on advanced molecular engineering techniques (directed
evolution725) to provide paramagnetic metalloprotein-based molecular MRI probes for
neuroimaging.726–727 In this approach, sophisticated protein engineering gave a
paramagnetic heme iron-containing probe that is selective for the neurotransmitter

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dopamine. Specific binding of dopamine to a side that is close to the heme iron altered T1-
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weighted MRI signals. Using this neurotransmitter sensing technique, the authors were able
to quantitatively map neurotransmitter release patterns deep within the living brain. The
chief limitation of the technology as it now stands is its relative insensitivity - only
neurotransmitter concentrations in excess of 2 µM could be detected.

A crown ether appended Gd(III)-complex capable of sensing amino acid neurotransmitters

was reported by Toth, Angelovski, and coworkers (Figure 89).728 The complex offers ditopic
binding for zwitterionic neurotransmitters via interactions (a) between the positively charged
and coordinatively unsaturated Gd(III) ion and the carboxylate function and (b) between a
pendant triazacrown ether and the amine function of the neurotransmitter. Relaxometric
titrations with different neurotransmitters, like γ-aminobutyric acid or glutamate remarkably
decreased the T1 relaxivity of their agent. The T1 relaxivity change can be attributed to a
switch in hydration state from q = 1.2 to q = 0.4. This complex was successfully employed
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to monitor neural activity in acute mouse brain slices by MRI.

6.7.2 Temperature-responsive MRI contrast agents—Recently, noninvasive

temperature monitoring with MRI has attracted attention since thermotherapy is rapidly
emerging. To this end, Zheng and coworkers developed a thermosensitive microgel based on
a manganese porphyrin core incorporated in a cross-linked poly(N-isopropylacrylamide)
material (Figure 90).729 Subtle temperature changes rapidly swell or shrink those microgels,
i.e. the volume of the microgel is changed with its lower critical solution temperature
(LCST, 29 – 33 °C). As the properties of the microgel change, the embedded paramagnetic
complexes experience changes in their rotational motion, which in turn influences r1.
Consequently, within a few degrees of temperature variation, an increase in r1 of 73 % could
be observed.
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Another concept utilizes thermosensitive liposomes that release a Gd-HPDO3A when heated
up.730 As a result the water-solubility of the Gd(III)-agent is increased, which in turn
decreases its T1 relaxivity.

Since chemical exchange rates are dependent on the temperature, ParaCEST agents have
also been used to generate temperature-dependent CEST effects.605, 731–733

It was also demonstrated that Fe(II) or Co(II)-based ParaSHIFT agents might be promising
candidates for MR thermometry applications.734–735

6.7.3 Light-responsive MRI contrast agents—The most prominent example for a

light-responsive MRI contrast agent couple is merocyanine-Gd-DO3A / spirooxazine-Gd-
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DO3A, which is also sensitive to the redox environment and was discussed above (Figure
78A). Following the same mechanism as discussed above, r1 is decreased after irradiation
with visible light at 563 nm and can be increased again by irradiation with UV light,
respectively.670–671

This concept was further applied for the development of a light-responsive T2* contrast
agent.736 Louie and coworkers coupled a spyropyran derivative to dextran sulfate coated iron
nanoparticles. Light-induced conformational changes of spyropyran between its

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 Wahsner et al. Page 85

hydrophopic and hydrophilic isomer led to aggregation or dispersion of the nanoparticles,

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respectively (Figure 91). Visible light induced aggregation of the particles increased their
superparamagnetism and hence, their T2* relaxivity.

6.7.4 DNA-responsive MRI contrast agents—Detecting specific nucleic acid

sequences within the in vitro and in vivo context is extremely challenging using MRI. This
is due to the extremely low concentrations of DNA, which is generally far below 1 µM. MRI
contrast agents have been designed that are nonspecific to a DNA sequence to be able to
track many types of gene delivery systems which can carry a high payload of DNA above
the MRI detection threshold. It was shown, that the CEST effect of a polymeric Eu(III)-
based ParaCEST agent could be influenced by interaction of the compound with DNA at 1
mM monomer concentration.737

In a different approach, magnetite spheres with DNA intercalators were bound to DNA
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duplexes (0.5 mM base pair concentration) for nucleic acid detection with a T2* MRI
contrast agent.738 However, the extreme aggregation of the iron oxide nanoparticles reduced
the water accessibility and therefore the superparamagnetism of the system, so that the
expected decrease of T2* was reversed in this example.

Summaries of the T1- and CEST activatable probes are listed in Tables 11 and 12,
respectively.

7. Activation and Retention

An effective strategy to overcome major challenges to achieving biochemically targeted or
activated MRI contrast is to combine the two approaches. There are multiple advantages to
combining activation and retention. For example, off target contrast enhancement can be
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avoided by developing a non-targeted “pro-agent” that is enzymatically activated to

accumulate at a tissue or cell target. Another advantage is that the relatively weak relaxivity
changes achieved with most activatable agents can be overcome if the activated agent is
accumulated in the aberrant microenvironment after washout of the unactivated agent.

The activation and retention strategy has been applied to image acute inflammation using the
myeloperoxidase sensing agent Gd-MPO, comprised of Gd-DTPA with two appended 5-
hydroxytryptamines.739–740 Myeloperoxidase is secreted by activated neutrophils and serves
to catalytically amplify the reactivity of reactive oxygen species released during neutrophil
burst by converting hydrogen peroxide into electrophilic forms of oxygen such as ferryl
heme and hypochlorouos acid. Myeloperoxidase mediated oxidation of Gd-MPO results in
5-hydroxytryptamine-based radicals that oligomerize to multimeric MPO and form covalent
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bonds to surface exposed tyrosines in nearby proteins. Self-oligomerization of Gd-MPO

does lead to relaxivity increase at field strenghts < 3.0 T but the strong, delayed
enhancement Gd-MPO provides at the inflammatory microenvironment is also due to
retention of the oxidized agent. Gd-MPO has been used to image the acute cardiovascular
inflammation in mouse models of myocardial infarction, stroke, and vasculitis, and in a
rabbit model of aneurism.741–744 Gd-MPO has also been used to image neuroinflammation
in a mouse model of multiple sclerosis.745

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 Wahsner et al. Page 86

Figure 92 shows Gd-MPO enhanced imaging of myeloperoxidase activity in a mouse model

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of myocardial infarction. The images in Figures 92A-C demonstrate the capability of Gd-
MPO to detect differences in myeloperoxidase activity in the infarct bearing hearts of wild
type, heterozygous, and homozygous myeloperoxidase knockout mice, respectively. The
infarct zone (solid yellow arrows) vs remote myocardium (open yellow block arrows) CNR
is correlated tightly with MPO activity, Figure 92D.

The activation and retention strategy was also applied to a EP-2104R derived agent designed
to differentiate active thrombosis from stable clot.746 The cysteine-disulfide bridge of
EP-2104R is key to fibrin affinity, and binding affinity is obviated upon breaking this bridge
to form open chain, mixed disulfides. An open chain, mixed disulfide form of EP-2104R
was converted back to EP-2104R via action of protein disulfide isomerase (PDI) enzymes
which are highly expressed and active on the surface of activated platelets. The mixed
disfulfide pro-agent was shown provide strong enhancement of clotted fibrin in the presence
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of PDI using in vitro phantoms. No clot enhancement was observed in control samples that
did not contain PDI.746

Neutrally charged and cell membrane permeant Mn-porphyrin and Mn-PDA agents have
been functionalized with acetoxymethyl esters that are cleaved by cytosolic estarases to
yield anionic, membrane impermeable carboxylate functionalized complexes.380, 747 These
intracellularly activated agents have been considered in cell labelling applications. For
example, the tetra-acetoxymethyl functionalized complex Mn-AMP was used to label mouse
embryonic cells. The Mn-AMP labelled cells remained strongly T1-enhanced as the cells
differentiated to cardiomyoctes in vitro.748 this strategy can ostensibly be extended to image
cell populations expressing high levels of myriad other forms of intracellular enzymatic
activities.
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The activation and retention approach has been applied to develop contrast agents that
accumulate in the microenvironments with high activities of matrix metalloprotease (MMP)
enzymes.626–628 These water soluble agents comprise Gd-DOTA conjugated to PEGylated
peptides that are proteolytically cleaved to yield hydrophobic peptides and thus a contrast
agent of poor solubility. This approach has been used to image MMP expression in various
mouse xenograft models of cancer. For example, an MMP-7 reactive agent was shown to
provide strong contrast enhancement of human colon cancer xenografts but strongly
attenuated contrast in mice treated with MMP inhibitors.626 In another study, an MMP-2
reactive agent provided strong tumor enhancement in a mouse model of breast cancer.627
The MMP-2 reactive agent was also capable of differentiating levels of MMP-2 expression
between two different mouse models of cancer.628
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8. Cell Tracking
A potentially highly impactful application of MRI contrast agents is in cell tracking.
Labelling exogenously implanted or endogenously occurring cells with an MRI contrast
agent enables non-invasive longitudinal tracking of cell mobility, survival, and
differentiation in vivo.

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One very promising area for MR cell tracking technology is tracking the fate of implanted
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cells during transplant or regenerative therapy. For example, ferucarbotran-labelled cells

were used to confirm successful transplantation and survival of β-cell containing pancreatic
islets in a rat model of Type I diabetes.749 The islet cells were isolated from healthy rats and
were labelled by incubation in ferucarbotran containing cell culture. The pancreatic islets
remained conspicuously hypointense for beyond 4 weeks after transplantation. Normal
blood sugar levels were obtained in the diabetic rats within 1 week of islet implantation.
This technology was extended to clinical trials performed on islet transplantation patients.
750–751 Injection of the contrast agent labelled cells was safe in all patients. Figures 93

shows the liver of a Type I diabetes patient receiving ferucarbotran labelled pancreatic islet
transplant via portal vein injection. Figures 93A-E were acquired prior to, 1 day, 1 week, 4
weeks, and 28 weeks after transplantation, respectively. The dark spots of signal loss are due
to ferucarbotran loaded islets (white arrows). MR cell tracking demonstrates that 60% of the
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implanted islets do not survive 1 week past engraftment injection, but most of the surviving
islets persist out to 24 weeks. The patients in this trial exhibited an increase in C-peptide
levels, a biomarker of endogenous insulin production, and 50-80% less insulin was required
to achieve near normal blood sugar levels.751

Islet transplantation has also been tracked by labelling with perfluorocarbon nanoparticles
and F-19 MRI.752 This direct F-19 detection strategy offers the advantage of quantitative
islet tracking with zero background interference.

MR cell tracking is also used to image the homing of immune cells in immunotherapy. In a
clinical trial of autologous dendritic cell therapy, immature dendritic cells isolated from the
blood were labelled with ferumoxides.753 Immature dendritic cells are phagocytic and
readily internalized the SPIONs. The ferumoxide-labelled dendritic cells were injected into a
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lymph node and migration to and accumulation in other lymph nodes was tracked by MRI.
MR tracking of the dendritic cells enabled retrospective identification of false negatives in
the clinical trial due to a high rate of mis-injection during intranodular implantation.753
Immunotherapy cell tracking has also been performed used F-19 MRI. In a clinical trial of
stage 4 colorectal cancer immunotherapy, dendritic cells isolated from the patient were
combined with lysate of surgically resected piece of the tumor in order to induce
presentation of tumor specific antigens, and incubated with perfuorocabon nanoparticles.
The F-19 labelled cells were administered intradermally and diminishment of the F-19
signal, due either to cell migration or to cell death and dispersion of the F-19 nanoparticles,
was monitored over the course of 24 h.10

Immune cells can also be labelled in vivo via a technique called magneto-transfection. Here,
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vaccine-contrast agent conjugates are injected which enables visualization of adaptive

immune system activation. The process by which antigen-presenting dendritic cells localize
in lymph nodes and initiate immune response has been monitored using MRI in a tumor
vaccine mouse model following injection of a SPION labelled vaccine.754

There are a number of examples where MR cell tracking has been used to monitor the fate of
stem cells during cell therapy. In a clinical trial of nerve regeneration in patients suffering
traumatic brain injury, autologous neural stem cells isolated from surgically excised tissue

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where labelled with ferumoxides and reinjected adjacent to the injury and cell migration to
the injured tissue was monitored over the course of 3 weeks.755 In another clinical study,
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autologous CD34+ marrow stem cells were labelled with magnetic beads. Labelling was
achieved by conjugating the magnetic beads to antibodies specific to CD34 antigen. The
labelled stem cells were injected intrathecally and cell migration and accumulation within
the lesion was observed over the course of 24 weeks.756 Cells have also been tracked via
labelling with CEST contrast agents. Immortalized mouse skeletal myoblasts were labelled
with Eu-HP-DO3A via hypertonic swelling of the cells and used for imaging in a mouse
model of cardiac cell therapy.757 The Eu-HP-DO3A labelled cells implanted in the wall of
the left ventricle of the heart provided over 30-fold greater MTRasym than surrounding tissue
or unlabeled cells immediately after injection. Cell survival and cell rejection were modelled
by implanting cells in in both syngeneic and allogeneic mouse models. In the syngeneic
mice the cells provided strong CEST contrast out to 20 days past injection, whereas the cells
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were rejected and no contrast was observed within 20 days of implantation in the allogenic
mice.

Cell tracking has also been used to monitor the dynamics of acute inflammation following
tissue injury. The time course of monocyte infiltration into the site of myocardial infarction
was visualized using T1-weighted MRI following injection of Gd-DTPA loaded liposomes.
554 Here, monocyte labelling was performed in vivo by exploiting the phagocytic nature of

monocytes. The time course of R1 enhancement in the infarcted myocardium correlated

strongly with ex vivo histologic measures of monocyte infiltration. A similar approach was
taken to image monocyte infiltration into tumors in a mouse model using F-19
perfluorocarbon nanoparticles.758 Here too, the particles were administered intravenously
and accumulated in monocytes by endogenous mechanisms. Tumor F-19 signal intensity
correlated with histologic counting of monocytes. In another study, analogously labelled
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F-19 monocytes were also used to image inflammation in a rat model of multiple sclerosis.
759 The F-19 signal was used to quantitatively differentiate relative macrophage burden on

the spinal cord of MS rats, MS rats receiving daily prophylactic cyclophosphamide treatment
(an immunosuppressant), and healthy control rats.

Cells can also be engineered to express reporter genes that provide endogenous MRI
contrast. In one proof of concept study, mice were treated intranasally with adenoviral vector
to deliver a reporter gene that results in expression of a ferritin protein engineered to localize
in the cytoplasm.760 Intranasal administration of the reporter gene carrying viral vector
resulted in strong T1-contrast of epithelial olfactory neurons. No contrast enhancement was
observed upon treatment with the same vector carrying only a green fluorescent protein
reporter gene. In another study the oncovirus G47Δ, which is currently being evaluated in
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humans for treatment of progressive glioblastoma, was engineered to carry a lysine rich
protein (LRP) CEST reporter and oncovirus transduction was imaged in a rat model of
glioma.761 Figures 94A-D show CEST MTRassym maps overlaid with T2-weighted images
of the glioma bearing brain prior to and 8h after intratumoral injection G47Δ carrying the
LRP reporter and LRP empty control virus. A >1% increase in magnetization MTRassym was
observed within 8 hours whereas no change in CEST contrast was observed after injection of
LRP-empty control, Figures 94E-F. LRP expression did not demonstrate any measureable
effect on the efficacy of the oncolytic viral therapy.

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There are also reporter genes that do not provide contrast but can lead to expression of
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receptors that can concentrate exogenously administered contrast agents within the cell, or
amplify the signal by reaction with activatable MRI contrast agents. Some of these strategies
have been described in previous sections. For example, the OATP seeking agent Gd-EOB-
DTPA has been used to track the fate of OATP expressing cells in vivo.526 Transduction of
the lacZ operon was confirmed using the β-galactosidase activated contrast agent.566 The
divalent metal ion transporter (DMT1), which accumulates Mn(II) with high affinity has
been used as an MR gene reporter.762 The DMT1 DNA construct was cloned into a viral
vector for transduction into murine glioma cells and intracranially implanted into mice. The
resultant DMT1 positive tumors were strongly enhanced in a T1-weighted scan 24h after
MnCl2 injection, whereas DMT negative control tumors were not T1 enhanced.

9. Conclusions
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The progress in MRI contrast agents has been mixed. When one of us co-wrote a review on
the topic in 1999 there had been several contrast agents approved for use in the preceding
decade and there were a number of new agents in clinical development. However in the
intervening time very few new agents were approved and many products, like the iron oxide
nanoparticles, have been discontinued because of low utilization rates. The lack of a
commercially successful new contrast agent has had a chilling effect on the clinical
development of new agents. Private investors are reluctant to spend tens of millions of
dollars to fund the clinical development of new imaging agents unless they believe that
future sales of the agent will be a multiple of that investment. A successful new contrast
agent must address a large market size and deliver useful diagnostic information that
justifies the cost of the contrast agent and the imaging procedure. A further barrier to
commercialization is the difficulty in obtain proof of concept data in humans. Unlike
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diagnostic radiopharmaceuticals which are given at microscopic doses, contrast agents are
administered in gram quantities necessitating kilo manufacturing and rigorous preclinical
safety studies before clinical trials can commence. Together this represents a few million
dollars of investment before human studies can begin.

On the other hand the need for specific molecular information is growing. We are entering
into a new era of precision medicine where treatments are tailored to the individual. New
powerful, but expensive, therapies are being developed, but many of these therapies are only
effective in a subset of patients. Increasingly, noninvasive methods like imaging are being
sought to stratify patients to therapies that will benefit them and avoid ineffective and/or
costly treatments. In 1999 we reported the first examples of activatable and targeted probes,
but now there are numerous examples some of which are well validated in animal models.
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Some of the targeted or activatable probes described here may find utility in the context of
precision medicine.

Fifteen years ago the safety of the commercial gadolinium(III) chelates was almost
unquestioned. But the advent of NSF and brain deposition has led to increased regulatory
scrutiny. We believe that there is a strong need for improved contrast agents that are less
retained, provide higher signal, and/or are more specific. It will be interesting to watch this

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field continue to evolve as chemists, biologists, physicists, engineers, and physicians

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continue to innovate.

Acknowledgments
J.W. thanks the German Research Foundation (DFG, research fellowship 320225462) for support. E.M.G
acknowledges grant support from the National Heart Lung and Blood Institute (K25HL128899, U54HL119145)
and the National Institute for Biomedical Imaging and Bioengineering (NIBIB, R21EB022804). P.C. is grateful for
funding from NIBIB (R01EB009062, xR21EB009738), the National Cancer Institute (R01CA161221), the National
Institute for Diabetes and Digestive and Kidney Diseases (U01104302), the National Instititute for Neurological
Diseases and Stroke (R01NS091552) as well as to Pfizer, Sanofi, and Siemens for supporting our work on MRI
contrast agents over the last decade. Julianne Johnson is gratefully acknowledged for her editorial and
administrative contributions. Dr. Pauline Désogère is warmly acknowledged for many helpful discussions. Patrick
Sheedy is thanked for his help with table 11 and table 12.

Biographies
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12. Author information

Jessica Wahsner is a post-doctoral fellow at the A. A. Martinos Center for Biomedical

Imaging, Massachusetts General Hospital and Harvard Medical School. She received both
her M.Sc. (2011) and Ph.D. (2015) in chemistry at the Ruhr-University Bochum, Germany,
under the mentorship of Prof. Michael Seitz. Receiving a fellowship by the German
Research Foundation (DFG), she started her current position as part of Prof. Peter Caravan’s
group in 2016. Her research interests primarily focus on the development of small molecule
PET probes for the molecular imaging of fibrotic disease and of bimodal PET-MR agents for
quantitative pH imaging.

Eric M. Gale is an Assistant Professor in Radiology at the A. A. Martinos Center for

Biomedical Imaging, Massachusetts General Hospital and Harvard Medical School. He
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received his B.A. in Chemistry from Rutgers University in 2006 and his Ph.D. in chemistry
from the University of Georgia under the mentorship of Todd Harrop, where he synthesized
and studied the reactivity of Ni complexes that model the nickel superoxide dismutase active
site. He received his postdoctoral training under the mentorship of Peter Caravan between
2012-2015, where he primarily focused on studying the chemistry of Mn complexes in the
context of MRI. He joined the faculty in 2015. His current research interests center around
developing Gd-free alternatives for commercially available contrast agents and on
developing redox active transition metal complexes for molecular MR imaging. He is a co-
founder of Reveal Pharmaceuticals.

Aurora Rodriguez-Rodriguez was a postdoctoral researcher for two years (2016-2017) at the
Athinoula A. Martinos Center for Biomedical Imaging at the Massachusetts General
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Hospital and Harvard Medical School, working in the group of Prof. Peter Caravan. She
received her Bachelor´s Degree in Chemistry (2008), her Master´s Degree in Environmental
and Fundamental Chemistry (2009) and her Ph.D. in Chemistry (2014) from the University
of A Coruña (Spain), under the supervision of Prof. María Teresa Rodríguez Blas and Dr.
Carlos Platas Iglesias. She was awarded with a 12 months postdoctoral fellowship from the
Conseil Général du Finistère 29 (France) in 2014 to work in the group ChASaM at the
University of Brittany (France) under the mentorship of Prof. Raphaël Tripier and Dr.

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Véronique Patinec. Her research is focus on the design, synthesis, characterization and
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application of new contrast agents for molecular imaging.

Peter Caravan is a native of Bay Roberts, Newfoundland, Canada. He received a B.Sc.

(Hons) from Acadia University in Nova Scotia and a Ph.D. in Inorganic Chemistry at the
University of British Columbia, where he was a National Sciences and Engineering
Research Council (NSERC) post-graduate scholar, under the mentorship of Professor Chris
Orvig. He was a NSERC post-doctoral fellow at the Université de Lausanne, Switzerland
where we worked with Professor André Merbach. He then joined Epix Pharmaceuticals in
Cambridge, MA where he worked on the development of targeted MRI contrast agents
including the FDA-approved blood pool agent gadofosveset (MS-325). In 2007 he was
recruited to establish a translational molecular imaging lab at the A. A. Martinos Center for
Biomedical Imaging at Massachusetts General Hospital and Harvard Medical School. In
2014 he was appointed Co-Director of the Institute for Innovation in Imaging (I3) which
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focuses on the clinical translation and commercialization of new radiological technologies

including contrast agents. He is a National Institutes of Health funded researcher with a
focus of development, application, and clinical translation of novel molecular MRI and PET
probes.

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Figure 1:
Route of administration of MRI contrast agents.
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Figure 2:
Main distribution sites and excretion pathways for intravenously administered soluble metal
complexes.15
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Figure 3:
Commercially approved T1 contrast agents (NMG = meglumine).
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Figure 4:
Chemical structures of the Gd(III)-based blood pool contrast agents (charges and counter
ions are omitted for simplicity).
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Figure 5:
Longitudinal (left) and transverse (right) relaxivities (mM−1s−1) of some commercial
gadolinium(III)-based contrast agents at different magnetic fields (green = 0.47 T, red = 1.5
T, blue = 3 T) in human plasma at 37 ºC.59
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Figure 6:
Paramagnetic contribution of 1 mM of T1 contrast agent Gd-DOTA on 1/T1 and 1/T2 in
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cortical grey matter at 1.5 T.

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Figure 7:
Axial T1-weighted MR images obtained at 3 T before (left) and 20 minutes after intravenous
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gadoteridol administration (right) of patient with glioblastoma. Reproduced with permission

of Ref.61 (URL: https://pubs.rsna.org/doi/abs/10.1148/radiol.12111472). Copyright 2013
Radiological Society of North America (RSNA®).
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Figure 8:
Paramagnetic contribution of 1 mM of T2 contrast agent ferucarbotran on 1/T1 and 1/T2 in
cortical grey matter at 1.5 T.
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Figure 9:
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T2-weighted images obtained before and 24 hours after intravenous ferumoxytol

administration show deep white matter lesions, demonstrated through several areas of
confluent, focal, strong signal loss due to ferumoxytol uptake (arrows). Reproduced with
permission of Ref.68 (URL: http://n.neurology.org/content/neurology/81/3/256). Copyright
2013 Wolters Kluwer Health, Inc.
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Figure 10:
CEST agents that are approved for use in clinical trials: glucose and iopamidol.70–72
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Figure 11:
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Chemical structures of some PFCs: left, hexafluorobenzene (HFB); center, perfluoro-15-

crown-5-ether (PFCE); right, tetra(perfluorotertbutyl)pentaerythritol (PERFECTA).
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Figure 12:
Examples of hydrophilic fluorinated molecules studied by Annapragada and coworkers.76
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Figure 13:
Examples of fluorinated compounds studied by Parker and coworkers in order to boost the
19FMRI – SNR.83–84
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Figure 14:
Change in the longitudinal relaxation time (a) and in the relaxivity (b) as a function of the
concentration of contrast agent in three different tissues: heart (T10 = 1200 ms), liver (T10 =
590 ms) and subcutaneous fat (T10 = 340 ms); supposing in every case r1 = 4 mM−1s−1.
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Figure 15:
Effect of field strength on relaxivity and contrast in the case of Gd-DOTA (left) and MS-325
(right) in white matter (WM) and grey matter (GM).
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Figure 16:
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Pictorial description of the parameters that influence the relaxivity of a MRI contrast agent.

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Figure 17:
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Gadolinium(III) complexes studied by 1H ENDOR where the Gd-H(water) distance was

determined.
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Figure 18:
Experimental 1H NMRD profiles showing relaxivity (per Gd(III)) versus field strength for
Gd-DTPA derivatives with short (A, MS-325), intermediate (B, EP-1084), and long (C,
MS-325 in HSA solution) rotational correlation times.
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Figure 19:
Effect of water residency time (τm, ns) for an inner-sphere water on r1 (—) and r2 (---) under
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optimal rotational conditions (τR, ns) at 0.47 T (A, 20 ns), 3 T (B, 0.5 ns), and 9.4 T (C, 0.5
ns). The optimal τm range increases as field increases. Reproduced with permission from
Ref.144 (URL: http://dx.doi.org/10.1002/cmmi.267). Copyright 2009 John Wiley and Sons.
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Figure 20:
TSAP and SAP isomers of DOTA-type ligands of macrocyclic lanthanide chelates.
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Figure 21:
Chirality sources within DOTA-like macrocyclic structures.
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Figure 22:
Ligands forming HSA-binding Gd(III) complexes without any water molecules in the inner-
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sphere (q = 0).17
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Figure 23:
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(a) Illustration of a CEST process: exchangable amine protons of a CEST agent are
selectively saturated using radio frequency (RF) irradiation, leading to a reduction of the
amine 1H signal intensity. Because of chemical exchange, the saturated protons (shown as
blue spheres) are transferred to the bulk water pool. Continous chemical exchange during the
saturation experiment leads to the amplification of the water signal reduction. (b)
Continuous application of RF pulses leads to the saturation of more bulk water protons,
thereby decreasing the 1H-NMR signal. (c) Z-spectrum (top): normalized water intensity

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(I/I0) vs off-resonance frequency (ΔRF), taking as 0.0 ppm the water resonance and the
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magnetization transfer ratio asymmetry (MTRasym) (ΔCS = separation in chemical shift

between the two proton pools) spectrum (bottom): which shows the Z-spectrum asymmetry
vs off-resonance frequency (ΔRF).180–181
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Figure 24:
Exogenous agents (blue) and endogenous biomolecules (red) that can be detected when
applying a saturation pulse at the magnetic resonance frequencies listed in the chart. (A)
Aryl acid agents, (B) aryl amide agents, (C) amides, (D) glutamate, (E) amines, (F) glucose,
and (G) lactic acid.189 Paramagnetic compounds have much wider chemical shift ranges.
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Figure 25:
Simplified scheme of a liposome (A) and a lipoCEST agent (B). 1H NMR spectra focused
on the region of the water signal, in a regular liposome (C) and in a lipoCEST agent (D).
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Figure 26:
Ligand systems: PCTA, AAZTA, CyPic3A, aDO3A and tacn(1-Me-3,2-hopo)3 that form
thermodynamically stable Gd(III)-complexes with extended hydration sphere.
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Figure 27:
Left: Structure of P03277; Right: Axial T1-weigthed images of the hepatic metastasis A)
before and B) after intravenous injection of 0.1 mmol kg−1 gadobutrol,; C) before and D)
after intravenous injection of 0.1 mmol kg−1 P03277. Arrow shows tumor. Reproduced with
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permission from Ref.288 (URL: https://journals.lww.com/10.1097/RLI.0000000000000192).

Copyright 2015 Wolters Kluwer Health, Inc.
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Figure 28:
Left: Plot of r1 (0.5 T and 25 °C) for monohydrated DOTA-based Gd(III) complexes vs.
molecular weight (R = 0.984). Right: Plot of τR evaluated from the NMRD profiles, vs.
molecular weight (R = 0.991). Reproduced with permission from Ref.289 (URL http://
dx.doi.org/10.1039/9781788010146-00121). Copyright 2018 Royal Society of Chemistry.
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Figure 29:
Chemical structure of the chelators mentioned in Figure 28.289
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Figure 30:
Different strategies to increase relaxivity by modulating rotational motion: A) linear polymer
of Gd(III)-complexes that rotates anisotropic (low relaxivity); B) Gd(III) chelates assembled
to a dendrimer that rotates isotropically, but internal motion is still possible (higher
relaxivity); C) Gd(III)-complex at the barycenter of the molecule, so that it can only rotate at
the rate of the entire molecule (highest relaxivity).109
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Figure 31:
Restricting the water ligand mobility (specifically rotation about the Gd-O bond), in Gd(III)
complexes through interaction with an intramolecular H-bond acceptor. Reproduced with
permission from Ref.123 (URL: http://dx.doi.org/10.1002/anie.201702274). Copyright 2017
John Wiley and Sons.
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Figure 32:
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Simulation illustrating the effect of internal motion on the field dependent T1 relaxivity
(hypothetical Gd(III)-compound with a global correlation time of 10 ns and a local
correlation time with 0.1 ns. F gives the degree of internal motion.109

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Figure 33:
Strategies for increasing relaxivity: A) Gd(III)-complex with targeting moiety that tumbles
fast (low relaxivity); B) Binding of the Gd(III)-complex to the targeting protein slows
tumbling and the relaxivity increases; C) Gd(III)-based multimer bound to the target protein,
relaxivity might be limited through internal motion; D) Gd(III)-based multimer bound to the
target protein via two points of attachment, limited internal motion increases relaxivity; E)
Structure of the Gd-DTPA-based multimer. Reproduced with permission from Ref.321
(URL: http://dx.doi.org/10.1002/anie.200502245). Copyright 2005 John Wiley and Sons.
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Figure 34:
T1 relaxivities of Gd3L3 (sphere), MS-325 with excess HSA (triangle) and Gd-HPDO3A
(diamond) as a function of magnetic field strength at 37 °C. A) T1 relaxivity plotted per
molecule, at 60 MHz and higher frequency: molecules with intermediate correlation time
(Gd3L3) are much more potent relaxation agents than slow (MS-325 with excess HSA) or
fast Gd-HPDO3A tumbling compounds; B) T1 relaxivity plotted per Gd(III)-ion shows the
same trend. Reproduced from Ref.324 (URL: http://dx.doi.org/10.1021/ja309187m).
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Copyright 2012 American Chemical Society.

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Figure 35:
Chemical structure of the Gd3L3.
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Figure 36:
Left: Effect of rotational correlation time on T1 relaxivity as a function of field for a Gd(III)
complex with a water residency time 100 ns. τR = 0.1 ns (…), 1.0 ns (---), 10 ns (—).
Reproduced with permission from Ref.144 (URL: http://dx.doi.org/10.1002/cmmi.267).
Copyright 2009 John Wiley and Sons. Right: Effect of HSA binding on T1 relaxation rate
for MS-325. Reproduced from Ref.17 (URL: http://dx.doi.org/10.1021/ja017168k).
Copyright 2002 American Chemical Society.
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Figure 37:
Relaxivity of 0.1 mM MS-325 and MS-325-BMA in pH 7.4 phosphate buffered saline or
0.67 mM HSA solution at 37 °C and 0.47 T. MS-325 is 88 % bound to HSA under these
conditions and MS-325-BMA is 83 % bound.109
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Figure 38:
Relaxivity of slow tumbling HSA-bound Gd-DOTA derivatives plotted vs. measured water
residency time at 37 °C at 20 MHz (filled triangles) or 60 MHz (open triangles),
respectively. τm limits relaxivity at a given field strength if it is either too short or too long.
The range of τm for optimal relaxivity becomes broader at higher field. Reproduced with
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permission from Ref.112 (URL: https://journals.lww.com/10.1097/RLI.0b013e3181ee5a9e).

Copyright 2010 Wolters Kluwer Health, Inc.

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Figure 39:
Chemical structures of Gd-EPTPA, Gd-DO3A-pyNox, Gd-DO3APABn, and Gd-ebpatcn.
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Figure 40:
NMRD relaxivity profiles of Gd-DOTA-NPs at different concentrations (T = 37 °C) showing
the characteristic shape of a compound with slow rotational correlation times. These results
indicate that the rotational motion of Gd-DOTA inside the hydrogel is restricted. Dotted line:
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NMRD relaxivity profile of Gd-DOTA (T = 37 °C). Reproduced with permission from Ref.
351 (URL: https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.201203190). Copyright 2012

John Wiley and Sons.

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Figure 41:
Thermodynamic stability of Gd-TRITA and Gd-TETA.353
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Figure 42:
Chemical structure of selected ligand systems.
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Figure 43:
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Mn(II) complexes previously considered in the context of MRI contrast.

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Figure 44:
Multiplanar reformatted coronal images from three-dimensional T1-weighted gradient echo
(volume-interpolated breathhold examination) sequence acquired at 3.0 T show abdominal
aorta and renal arteries, A, prior to injection of contrast agent, B, 9 seconds after injection of
0.1 mmol/kg Mn-PyC3A, and, C, 9 seconds after injection of 0.1 mmol/kg Gd-DTPA.
Reproduced with permission from Ref.378 (URL: https://pubs.rsna.org/doi/abs/10.1148/
radiol.2017170977). Copyright 2017 Radiological Society of North America (RSNA®).
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Figure 45:
Mn(III) complexes explored as high-relaxivity MRI contrast agents.
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Figure 46:
(A) The Fe(III) complexes Fe-HBED and Fe-EHPG and (B) have been considered as
hepatobiliary specific contrast agents. (B) The Fe(II) complexes Fe-DPTACN and Fe-
DTTACN have been considered as contrast agents.
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Figure 47:
Eu(II) complexes considered as potential MRI contrast agents.
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Figure 48:
The nitroxide radicals 3-CP and chex have been evaluated as MRI contrast agents.
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Figure 49:
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Iodinated X-ray contrast agents have been evaluated as DiaCEST contrast agents.
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Figure 50:
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The exchangeable amide protons of Dy-DOTAM provide a greater CEST effect than the
protons of the exchangeable water co-ligand because the amide protons are further from the
Dy(III) ion and thus experience a weaker paramagnetic relaxation effect.

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Figure 51:
A) Fibrin-targeting contrast agent EP-2104R is the only biochemically targeted agent to be
used to detect a pathologic biomarker in humans. B) Short axis view of the heart before
EP-2104R injection. The arrow indicates the location of a left ventricular thrombus that is
difficult to discern in the baseline scan. C) The thrombus is highly conspicuous for several
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hours after EP-2104R injection. Reproduced with permission from Ref.463 (URL: https://
link.springer.com/article/10.1007%2Fs00330-008-0965-2). Copyright 2008 Springer Nature.

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Figure 52:
The Mn-based fibrin-seeking contrast agent Mn-FBP is comprised of 4 Mn-PyC3A
chelators conjugated to a fibrin-binding peptide.
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Figure 53:
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A) Collagen-targeting MRI contrast agents are derived from a collagen-binding peptide

identified via high-throughput screen to possess high specificity and affinity for typeI
collagen. B,C) The collagen-targeted agents EP-3533 and CM-101 are comprised of the
collagen-binding peptide and 3 Gd-DTPA or Gd-DOTA chelators connected to the peptide
N-terminus and lysine ε-side chains via thiourea or amide linkages, respectively. C)
EP-3600 is comprised of the collagen-binding peptide and a trimeric Gd-DOTA containing
scaffold via the peptide N-terminus.

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Figure 54:
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Change in contrast-to-noise ratio (CNR) following CM-101 injection in bile duct ligated
(BDL, liver fibrosis) and sham operated (healthy non-fibrotic liver) rats. A) Representative
dynamic time courses of the change in CNR between liver and muscle tissue for BDL (red
dots) and sham (blue dots) treated rats following injection of CM-101. The maximal
difference in ΔCNR (green line) between sham operated and BDL rats was observed at
approximately 15-20 minutes following CM-101 injection. B) A statistically significant
difference in the area under the ΔCNR curve (AUC) between sham operated and BDL rats

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was observed. C) A statistically significant difference in ΔCNR between sham operated and
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BDL rats was also observed at 15 minutes post CM-101 injection. D) Representative T1-
weighted images acquired pre CM-101 (baseline) and 15 minutes post CM-101 injection.
Notice that the fibrotic liver is markedly enhanced with CM-101 but healthy liver is not.
Reproduced with permission from Ref.492 (URL: https://pubs.rsna.org/doi/10.1148/radiol.
2017170595). Copyright 2017 Radiological Society of North America (RSNA®).
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Figure 55:
A) Allysine-targeted agents Gd-Hyd and Gd-OA, and their corresponding non-targeted
control agents Gd-DiMe and Gd-OX. B-D) Imaging and ex vivo tissue analyses for Gd-OA
(allysine-targeting) an in naive (no fibrogenesis) and bleomycin (Bleo) injured (active
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fibrogenesis) mice. B) Quantification of the change in lung-to-muscle signal intensity ratio

following injection of Gd-OA to naïve and Bleo injured mice. C) Quantification of allysine
in naïve and Bleo injured mice. D) Coronal MR images of naïve and bleomycin injured mice
where the false color overlay is the difference image of the post Gd-OA image and the
baseline image. Reproduced with permission from Ref.500 (URL: https://
onlinelibrary.wiley.com/doi/abs/10.1002/anie.201704773). Copyright 2017 John Wiley and
Sons.
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Figure 56:
The CLT1 and CREKA peptides identified via high-throughput screen to bind to the fibrin-
fibronectic complex with high affinity. A) A fibronectin-targeting agents comprised of Gd-
DTPA conjugated to CLT1 via the peptide N-terminus and high payload fibronectin-
targeting agents comprising multimeric Gd(III)-complexes (R1 and R2) conjugated to the to
the CREKA peptide. B) One high-payload fibronectin-targeting agent was assembled by
conjugating a tetrameric Gd-DOTA scaffold to the CREKA N-terminus, C) another was
assembled via conjugation of a trimeric Gd-DOTA scaffold to the cysteine side chain S.
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Figure 57:
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(A) Elastin-targeting MRI contrast agent Gd-ESMA. (B-G) show MR imaging of the
suprarenal abdominal aorta in placebo treated mice (B-D) and mice bearing a
pharmacologically-induced abdominal aortic aneurysm (E-G). Time of flight MR
angiograms are shown in (B) and (E), the red lines mark the axial cross sections of the
vessels depicted in images (B-D) and (F-G). Images (C-D) and (F-G) were acquired using
the same T1-weighted protocol. (C) and (F) were acquired without contrast enhancement,
whereas (D) and (G) were contrast-enhanced using Gd-ESMA. The aneurysm bearing vessel

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is strongly enhanced compared to the vessel in the negative control animal because an
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increase in elastin content accompanies the compensatory remodeling of vessel wall at the
site of the dilation. Reproduced with permission from Ref.507 (URL: https://
www.ahajournals.org/doi/full/10.1161/CIRCIMAGING.113.001131). Copyright 2014
Wolters Kluwer Health, Inc.
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Figure 58:
Myelin targeting MRI contrast agent, Case Myelin Compound.
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Figure 59:
Comparison of (A) Gd-DOTA enhanced and (B) glucose enhanced imaging in a 45 year old
woman with glioblastoma, the Gd-DOTA and glucose enhanced images are fused in (C)
Glucose accumulates in proliferating cells and provides strong contrast enhancement in T1ρ-
weighted images. The glucose enhanced images provide strong signal intensity in the
dorsomedial regions of the tumor that overlap with T1-enhancement, but also strong
enhancement beyond the blood-brain barrier disruption. Reproduced with permission from
Ref.531 (URL: https://pubs.rsna.org/doi/10.1148/radiol.2017162351). Copyright 2017
Radiological Society of North America (RSNA®).
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Figure 60:
High-affinity folate receptor-targeting MRI contrast agent comprised of folate conjugated to
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Gd-DOTA via a bis(aminoethyl)glyocol linker.

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Figure 61:
Gd-DTPA-B(SLex)A is an E-selectin-targeting agent. This agent has been used to image
endothelial cell activation in a mouse model of fulminant hepatitis.
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Figure 62:
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Gd-TO binds to extracellular DNA via DNA intercalation of the appended thiazole orange
dye. This agent has been used to image necrosis in a mouse model of myocardial infarction
and to monitor the dynamics of extracellular DNA clearance after an acute necrotic event.

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Figure 63:
Detecting enzyme activity with a Gd(III)-based MR contrast agent through changes in T1
relaxation caused by modulation of inner-sphere hydration state. Chemical structures of the
first (A) and second (B) generation β-galactosidase sensors. C) Enzymatic cleavage of β-
galactose frees one coordination side that becomes accessible by water molecules. D) Two
living Xenopus laevis embryos were injected with the second generation of the β-
galactosidase sensor. The embryo shown on the right was also injected with β-galactosidase
mRNA. The pseudocolor rendering of MR images shows that the signal strength is 45 –
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65 % greater in the embryo treated with ß-galactosidase mRNA, demonstrating the detection
of β-galactosidase activity. Labeled anatomy: (e) eye, (c) cement gland, (s) somite, (b)
brachial arches. Reproduced with permission from Ref.566 (URL: http://dx.doi.org/
10.1038/73780). Copyright 2000 Springer Nature.
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Figure 64:
Selected pH-responsive T1 relaxivity agents.574,308, 577, 580–582
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Figure 65:
Selected pH-responsive T1 relaxivity agents.586–589
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Figure 66:
Selected pH-responsive DiaCEST agents.8, 449, 590–595
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Figure 67:
pH dependency of proton exchange and its relationship to CEST. Left: 1H-NMR spectra of
poly-L-lysine at different pH. Right: CEST spectra of poly-L-lysine at different pH. The
proton exchange accelerates with increasing pH, which broadens the corresponding 1H-
NMR signal but amplifies the CEST signal. Reproduced with permission from Ref.591
(URL: http://dx.doi.org/10.1002/mrm.20818). Copyright 2006 John Wiley and Sons.
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Figure 68:
Selected pH-responsive ParaCEST agents (protons employed for CEST imaging are shown
in red).600–606
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Figure 69:
T2W images (7 T) acquired pre (A) and 2 min post (D) i.v. injection of Yb-HPDO3A (dose:
1.2 mmol kg−1); STmaps calculated at 66 ppm pre (B) and 2 min post (E) i.v. injection of
Yb-HPDO3A; STmaps calculated at 92 ppm pre (C) and 2 min post (F) i.v. injection of Yb-
HPDO3A; G) pH map of a region of a tumor isolated in slice one calculated from the
corresponding ST maps. Reproduced with permission from Ref.611 (URL: http://dx.doi.org/
10.1002/mrm.24664). Copyright 2014 John Wiley and Sons.
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Figure 70:
First frequency-responsive transition-metal based ParaCEST agent that reports on pH.612
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Figure 71:
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pH-responsive ParaSHIFT agent.613

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Figure 72:
Selected enzyme-responsive T1 MRI contrast agents (enzyme cleavable entities are shown in
blue).623–625, 630
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Figure 73:
A) Chemical structure of D-DOTA(Gd); B) Interaction of Gd-DTPA-diTyr with activated
tyrosinase can result in: a) oligomers and b) contrast agent-albumin conjugates. Both
products yield an increase in relaxivity. Reproduced with permission from Ref.637 (URL:
http://dx.doi.org/10.1002/cbic.200700157). Copyright 2007 John Wiley and Sons. C)
Tyrosinase-responsive Mn(II) ligands.634
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Figure 74:
Design and mechanism of action of the caspase-3-sensitive nanoaggregation MRI probe (C-
SNAM). (left) Chemical structure of C-SNAM (1). Caspase-3 catalyzed DEVD peptide
cleavage and GSH mediated disulfide reduction triggers a biocompatible intramolecular
cyclization reaction that yields the rigid and hydrophobic macrocycle (2). Subsequent self-
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assembly to Gd(III) containing nanoparticles increases r1 by 90 %. (right) Mechanism of

action in vivo: (top) Intra-articular injection of C-SNAM into rat knee joints with implants of
apoptotic and viable stem cells. (bottom) Caspase-3 mediated activation leads to an
enhanced MRI signal through increased T1 relaxivity and retention in apoptotic stem cell
transplants. Reproduced from Ref.640 (URL: https://doi.org/10.1021/nn504494c). Copyright
2015 American Chemical Society.

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Figure 75:
Chemical structure of Gd-DOTA-DEVD-Tfb.651
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Figure 76:
Selected enzyme-responsive ParaCEST MRI contrast agents (enzyme cleavable entities are
shown in blue).655, 657, 659–660, 662
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Figure 77:
Selected enzyme-responsive DiaCEST MRI contrast agents (enzyme cleavable entities are
shown in blue).663, 665–669
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Figure 78:
Selected redox potential-responsive T1 contrast agents.382, 657, 670–673, 675–679, 686, 689
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Figure 79:
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Vascular volume and relative deoxygenation maps of a representative tumor. (A) Vascular
volume maps of 7 slices covering the tumor; (B) relative deoxygenation maps of 7 slices
covering the tumor; (C) magnification of vascular volume (top) and relative deoxygenation
(bottom) maps of the central slice of tumor; (D) T2w image of the central slice of tumor.
Reproduced from Ref.690 (URL: https://doi.org/10.1021/acsnano.5b02604). Copyright 2015
American Chemical Society.

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Figure 80:
Selected redox potential responsive ParaCEST agents.184, 201, 691–693
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Figure 81:
A) Schematic representation of the dual T1-CEST liposomal agent. The CEST contrast is
activated after cleavage of the Gd(III)-complexes by a biological trigger, e.g. reducing
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environment. B) MR images of a phantom at 7 T and 37 °C of 1) LipoCEST agent 2)

LipoCEST agent modified with free thiol groups on the surface 3) LipoCEST agent
modified witch Gd-DO3A derivative via disulfide linkage (LipoCEST-SS-DO3A) 4)
Treatment of LipoCEST-SS-DO3A with the reducing agent TCEP. Left: T1 weighted
images; right: CEST map upon radiation at 3.5 ppm overlaid on a T2-weighted image of the
phantom. Reproduced with permission from Ref.695 (URL: http://dx.doi.org/10.1039/
C1CC10172B). Copyright 2011 Royal Society of Chemistry.
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Figure 82:
Selected Zn(II) binding MRI contrast agents.696–699
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Figure 83:
A) Structure of Gd-CP027; B) Gd-CP027 binds to Zn(II) and forms a complex with HSA;
C) Glucose sensitive contrast enhanced (GSCE) T1 weighted MR images at 9.4 T of the
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prostate of mice during various stages of tumor development: (Bottom) image of a normal
healthy prostate (Middle) image of malignant prostate with WD tumor shows clear
hypointensity due to the presumable lack of intracellular Zn(II) (top) image of malignant
prostate with PD shows no GSCE D) Average GSCE measured over the entire prostate of
mice (WD tumor = well differentiated tumors, PD tumor = poorly differentiated tumor).
Reproduced from Ref.702 (URL: http://www.pnas.org/content/113/37/E5464). Copyright
2016 National Academy of Science.

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Figure 84:
Selected Zn(II) binding MRI contrast agents.703–704
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Figure 85:
A) Ca(II)-responsive MRI contrast agent706–707 B) Ca(II)-responsive MRI contrast agent
that can discriminate between extra- and intracellular Ca(II) ions.708
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Figure 86:
Selected Ca(II) binding MRI contrast agents.706–707, 709, 711
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Figure 87:
Schematic mechanism that causes the change in T1 relaxivity in the presence of Ca(II)-ions
of a liposomal Ca(II)-responsive Gd(III)-based contrast agent. Reproduced from Ref.714
(URL: https://doi.org/10.1021/acs.biomac.5b01668). Copyright 2016 American Chemical
Society.
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 Wahsner et al. Page 224
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Figure 88:
A) Schematic illustration of a copper binding Gd(III)-based probe that changes its hydration
state in the presence of copper ions. B) – E) Selected Cu(I)/Cu(II) binding MRI contrast
agents.716–719
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 Wahsner et al. Page 225
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Figure 89:
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A crown ether appended Gd(III)-complex capable of sensing zwitterionic amino acid

neurotransmitters. 728

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 Wahsner et al. Page 226
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Figure 90:
Schematic concept of thermosensitive microgels based on a manganese porphyrin core
incorporated in a cross-linked poly(N-isopropylacrylamide) material. LCST: lower critical
solution temperature 29 – 33 °C. Reproduced from Ref.729 (URL: https://doi.org/10.1021/
acsmacrolett.5b00058). Copyright 2015 American Chemical Society.
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 Wahsner et al. Page 227
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Figure 91:
Proposed mechanism of light-induced reversible aggregation of iron oxide nanoparticles
coupled to a spyropyran derivative. Reproduced from Ref.736 (URL: https://doi.org/10.1021/
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ja100254m). Copyright 2010 American Chemical Society.

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 Wahsner et al. Page 228
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Figure 92:
A-C) T1-weighted images showing short axis views of the infarct bearing hearts of wild
type, heterozygous, and homozygous myeloperoxidase knockout mice, respectively. The
myocardial infarction is donoted by the solid yellow arrows and the remote myocardium is
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denoted by the open yellow block arrows. D) Infract zone vs. remote myocardium CNR is
tightly correlated to myeloperoxidase activity within the infarction. Reproduced with
permission from Ref.742 (URL: https://www.ahajournals.org/doi/full/10.1161/
CIRCULATIONAHA.107.756510). Copyright 2008 Wolters Kluwer Health, Inc.
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 Wahsner et al. Page 229
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Figure 93:
Coronal view of the liver of a Type I diabetes patient receiving implantation of ferucarbotran
labelled pancreatic islets via portal vein injection. A-E) Images were acquired prior to, 1
day, 1 week, 4 weeks, and 28 weeks after transplantation, respectively. The images show that
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roughly 60% of the engrafted islet do not survive past 1 week after engraftment, but most of
the surviving islets persist out past 24 weeks. Reproduced with permission from Ref.751
(URL: https://doi.org/10.1097%2FTP.0b013e3181ffba5e). Copyright 2010 Wolters Kluwer
Health, Inc.
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 Wahsner et al. Page 230
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Figure 94:
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CEST MTRassym maps overlaid with T2-weighted images of glioma bearing rat brain prior
to and 8h after injection G47Δ carrying the LRP reporter and LRP empty control virus.
(A,B) Images recorded prior to and 8h after direct intratumoral injection of G47Δ carrying
the LRP reporter. (C,D) Images recorded prior to and 8h after direct intratumoral injection of
LRP empty G47Δ. (E) Comparison of MTRassym prior to and 8h after treatment with virus
carrying the LRP reporter gene and LRP empty control. (F) Comparison of ΔMTRassym
observed 8h after treatment with virus carrying the LRP reporter gene and LRP empty

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 Wahsner et al. Page 231

control. Reproduced with permission from Ref.761 (URL: https://pubs.rsna.org/doi/10.1148/

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radiol.14140251). Copyright 2015 Radiological Society of North America (RSNA®).

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 Wahsner et al. Page 232

Table 1:

ECF MRI contrast agents that have been used in the clinic.
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ECF agent (Trade name) ECF agent (Chemical code) ECF agent (Generic name) Approval date
Dotarem, Clariscan Gd-DOTA gadoterate meglumine 1989 (Europe)
2013 (United States)
ProHance Gd-HPDO3A gadoteridol 1992
Gadovist (Europe) Gd-DO3A-butrol gadobutrol 1998 (Europe)
Gadavist (United States) 2011 (United States)
a Gd-DTPA gadopentate dimeglumine 1988
Magnevist
a Gd-DTPA-BMA gadodiamide 1993
Omniscan
a Gd-DTPA-BMEA gadoversetamide 1999
Optimark
b, c Gd-BOPTA gadobenate dimeglumine 2004
Multihance

a
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agents suspended by the European Medicines Agency in 2017.

b
agent available for limited, liver-specific indications in the EU.
c
multipurpose agent that is also suitable for liver imaging.14
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 Wahsner et al. Page 233

Table 2:

Examples of blood pool contrast agents that have been used in human clinical trials.
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Blood pool agent (Trade name) Blood pool agent (Chemical code) Blood pool agent (Generic name) Approval date
Approved for use as an iron replacement therapy. Not approved, but used off-label for MRA
Feraheme ferumoxytol (USPIO) 2009 (United States)
2013 (Europe)

Approved for MRA but no longer commercially available:

Ablavar (formerly: Vasovist, Angiomark) MS-325 gadofosveset trisodium 2005 (Europe)

2008 (United States)

Used in clinical trials but clinical development of the agents has been discontinued:
B22956 Gadocoletic acid -
Gadomer SH L 643A Gadomer-17 -
Vistarem P792 Gadomelitol -
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Clariscan NC-100150 PEG-feron (USPIO) -

VSOP-C184 -
Sinerem/Combidex AMI-227 ferumoxtran-10 (USPIO) -
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 Wahsner et al. Page 234

Table 3:

Organ-specific MRI contrast agents that have been used clinically or received approval for clinical trials.
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Organ-specific agent Organ-specific Organ-specific agent Approval date Applications

(Trade name) agent (Short name) (Generic name)
Approved and commercially available:
Primovist (Europe) Gd-EOB-DTPA disodium gadoxetic acid 2005 (Europe) liver
Eovist (United States) 2008 (United States)
a Gd-BOPTA gadobenate dimeglumine 1998 (Europe) liver
Multihance 2004 (United States)

Approved for use as an iron replacement therapy. Not approved, but used off-label for MRA
Feraheme ferumoxytol (USPIO) 2009 (United States) brain lesions, abdominal organs,
2013 (Europe) lymph nodes, vascular walls

Agent withdrawn from one or all major markets:

Teslascan Mn-DPDP mangafodipir trisodium 1997 liver, myocardium
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Feridex (United States) AMI-25 ferumoxides (SPIO) 1996 (United States) liver
Endorem (Europe)

Resovist SH U 555 A ferucarbotran (SPIO) 2001 (Europe) liver

Used in clinical trials but clinical development of the agents has been discontinued:
Dy-DTPA-BMA sprodiamde injection - myocardium, brain perfusions
Sinerem/Combidex AMI-227 ferumoxtran-10 (USPIO) - metastatic lymph nodes,
macrophage imaging

a
Gd-BOPTA is predominantly used as an ECF agent but does provide some hepatobiliary enhancement and can be used for liver imaging
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 Wahsner et al. Page 235

Table 4:

OCAs that are approved for clinical application.

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OC agent (Trade name) OC agent (Short name) OC agent (Generic name) Approval date
Lumirem/GastroMARK AMI-121 ferumoxsil (MPIO) 1996
Ferriseltz - ferric ammonium citrate 1992
LumenHance - manganese chloride 1997
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 Wahsner et al. Page 236

Table 5:

Thermodynamic and conditional (pH 7.4) stability constants for commercially approved Gd(III)-based MRI
Author Manuscript

contrast agents.206

Charge Log KGdL Log Kcond

[Gd(DTPA)(H2O)]2− −2 22.46207 18.4

[Gd(DTPA-BMA)(H2O)] 0 16.85203 14.8

[Gd(DTPA-BMEA)(H2O)] 0 16.6208 15.0

[Gd(BOPTA)(H2 O)]2− −2 22.6208 18.4

[Gd(EOB-DTPA)(H2 O)]2− −2 23.46209 18.7

MS-325 −3 22.1210 18.9

[Gd(DOTA)(H2O)]− −1 24.7211 17.2

[Gd(HP-DO3A)(H2O)] 0 23.8208 17.1

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[Gd(DO3A-butrol)(H2O)] 0 21.8212 14.7

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 Wahsner et al. Page 237

Table 6:

Kinetic measures for dissociation of Gd(III) from commercially approved MRI contrast agents under different
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conditions.

Acid-catalyzed dissociation rate 1 eq. Zn, phosphate buffer pH 10 mM phosphate, human

constants219 7.0, 37 °C110, 217 plasma, 37 °C206, 220

k1 /M−1 s−1 t0.8/min

a
t2%/h
b

[Gd(DTPA)(H2O)]2− 0.58 260-280 8.73

[Gd(DTPA-BMA)(H2O)] 12.7 50-60 0.51

[Gd(DTPA-BMEA)(H2O)] 8.6 0.89

[Gd(BOPTA)(H2O)]2− 0.41 600 9.08

[Gd(EOB-DTPA)(H2O)]2− 0.16 1500 51.3

MS-325 2.9 × 10−2 3800 34.4

[Gd(DOTA)(H2 O)]− (8.4 × 10−6)221 >5000 > 1000

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(1.8 × 10−6)222
[Gd(HP-DO3A)(H2O)] (6.4 × 10−4)204 >5000 > 1000
(2.6 × 10−4)223
[Gd(DO3A-butrol)(H2O)] 2.8 × 10−5 >5000 > 1000

a
competition against Zn(II) - 2.5 mM Gd(III)-based contrast agent, 2.5 mM ZnCl2, pH = 7.0, 50 mM phosphate buffer (t0.8 = time for R1 to reach
80% of its initial relaxivity).
b
t2% = time when 2% of the Gd(III)-ions are released from the complex.
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 Wahsner et al. Page 238

Table 7:

Comparison of formation constants of previously reported Mn chelates. In each group, chelates are ordered by
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conditional log KML at pH 7.4.

log KML log KML pH 7.4 a

pMn

Acyclic Chelates, q < 1

AAZTA368 14.19
b
11.31
b
8.15
b

DTPA363 14.54
b
10.93
b
7.77
b

EGTA363 11.60
b
8.82
b
6.91
b

AAZ3A368 11.00
b
8.15
b
6.58
b

AAZ3MA368 10.67
b
7.77
b
6.39
b

BIMP363 9.74
b
7.57
b
6.30
b
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MeAAZ3A368 11.43
b
7.03
b
6.47
b

PMDPA369 11.37
f
6.82
f
5.94
f

TMDTA363 9.70
b
6.54
b
5.81
b

DPDP370 15.10
d
6.33
d
5.72
d

HBED370 14.78
d
6.33
d
5.72
d

PLED370 12.56
d
6.09
d
5.61
d

Acyclic Chelates, q ≥ 1
CyDTA363, 371 b
14.32 , 14.69
d b
12.34 , 12.64
d b
8.67 , 8.82
d

PyC3A b b c b
13.86 11.34 , 11.40 8.06
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EDTA-BOM2332 13.90
e
11.02
e
8.12
e

EDTA-BOM332 13.50
e
10.62
e
7.82
e

EDTA332, 363, 371 b d

12.46 , 12.61 , 13.88
e b d
10.49 , 10.67 , 11.02
e b d
7.82 , 7.83 , 8.01
e

CyHBET-NO2371 13.66
d
10.10
d
7.55
d

HBET-NO2371 11.29
d
8.73
d
7.01
d

HBET371 13.07
d
7.97
d
6.62
d

HBET-OMe371 13.32
d
7.91
d
6.48
d

CyHBET371 14.16
d
7.76
d
6.68
d

CyHBET-OMe371 14.61
d
6.95
d
6.24
d
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Macrocyclic Chelates, q < 1

DOTA372 19.89
g
13.27
g
8.94
g

DO3A372 19.49
g
12.25
g
8.62
g

HPDO3A372 17.89
g
12.18
g
8.59
g

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 Wahsner et al. Page 239

log KML log KML pH 7.4 a

pMn
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NOTA373-374 g
14.90 , 16.3
g g
10.88 , 10.52
g g
7.94 , 7.76
g

i g g g
1,4-DO2A 372 16.13 10.10 7.55
1,7-DO2A372 14.54
g
8.21
g
6.61
g

Macrocylic Chelators, q ≥ 1
15-pyN5367 10.89
g
7.72
g
6.37
g

12-pyN4P366 14.06
g
7.35
g
6.19
g

12-pyN4A366 11.54
g
7.14
g
6.09
g

NO2A365 11.56
h
7.11
h
5.96
h

NODAHA375 10.15
f
6.43
f
5.76
f

NODAHep 375 10.98

f
6.37
f
5.73
f
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NODABA375 9.90
f
6.10
f
5.61
f

15-pyN3O2367 7.18
g
5.20
g
5.27
g

9-aneN2O-2P365 10.61
g
5.07
g
5.23
g

9-aneN2O-2A365 7.43
g
4.36
g
5.06
g

9-aneN2O-2PH 365 4.30

g
3.34
g
5.01
g

9-aneN2O-2PPh 365 4.82

g
2.98
g
5.00
g

a
pMn at 10 µM MnL, pH 7.4;
b
I = 0.15 M NaCl, 25 °C;
c
determined from Kcomp with CDTA challenge, pH 7.4 Tris 50 mM, RT;
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d
I = 0.10 M NaCl, 25 °C;
e
I = 1.0 M KCl, 25 °C;
f
I = 0.1 M KCl, 25 °C;
g
I = 0.1 M Me4NCl, 25 °C;
h
I = 0.1 M NaNO3, 25 °C.
i
Mn-DO2A is 0 < q < 1.
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 Wahsner et al. Page 240

Table 8:

Comparison of r1, r2, hydration state (q), water exchange rate (kex), enthalpy of activation of water exchange
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(ΔH≠), and Mn-H(water) hyperfine coupling constant for Mn(II) complexes that have been considered as MRI
contrast agents.

r1298 (mM−1s−1) r1310 (mM−1s−1) r2310 (mM−1s−1) q kex310 (× 107 s−1) ΔH≠ Ao/ħ (× 107 rad/s)

PyC3A a b a b a 1 11 37.2 2.87

2.8 , 3.3 2.1 , 2.5 4.9
EDTA332 3.0
c
2.2
a
3.7
a 1 59 36.7 3.79

EDTA-BOM332 3.6
b -- -- 1 19 43.1 3.79

EDTA-BOM2332 4.3
b -- -- 1 25 38.4 3.79

EDTA-Tyr iso334 -- a
3.2 , 3.6
b 1 30 35.6 2.92

Mn-453138, 331 5.8

b -- -- 0.8 30 21.1 3.33
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CDTA138 --
2.1
a,d
3.5
a,d 1 27 35.8 3.14

AAZTA368 1.6
b368 -- -- 0 -- -- --

AAZ3A368 2.5
b -- -- 0<q<1 7.0 22.8 --

MeAAZ3A368 2.0
b -- -- 0<q<1 20 26.5 --

AAZ3MA368 1.9
b -- -- 0<q<1 18 16.7 --

HBET371 --
2.8
a
9.4
a 1 370 33.8 3.54

HBET-OMe371 --
3.1
a
11.1
a 1 360 40.7 4.15

HBET-NO2371 --
2.3
a
4.8
a 0.5 48 41.2 3.48

CyHBET371 --
3.3
a
6.0
a 1 770 41.2 3.36
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CyHBET-OMe371 --
3.3
a
5.8
a 1 300 20.7 4.02

CyHBET-NO2371 --
2.3
a
3.7
a 1 190 31.3 3.97

ENOTA376 3.4
b
2.7
b -- 1 7.9 20.5 3.27

9-aneN2O-2A365 2.8
b
2.3
b -- 1 2.3 38.8 3.33

9-aneN2O-2P365 5.1
b
4.3
b -- 1 140 11.7 3.33

1,4-DO2A364 2.1
b -- -- 0<q<1 110 29.4 4.10

1,7-DO2A364 1.5
b -- -- 0 -- -- --

DO1A364 2.4
b -- -- 1 500 17.6 3.94

12-pyN4A366 2.4
b
1.9
b -- 1 330 13.0 3.66
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12-pyN4P366 2.8
b
2.3
b -- 1 250 14.0 3.99

15-pyN5367 4.5
b
3.6
b -- 2 13 37.7 3.86

15-pyN3O2367 3.6
b
3.1
b -- 2 0.69 35.3 3.86

15-pyN5367 4.5
b
3.6
b -- 2 13 37.7 3.86

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a
1.4 T;
b
0.47 T;
c
0.56 T.
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Table 9:

Dissociation half-lives of Mn(II) complexes in the presence of competing divalent metal ions. Conditions: 10
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µM Mn(II) complex, 10 µM Cu(II) or Zn(II), pH 7.4.

ligand t1/2 (h)

a 0.08
EDTA

a 12
CyDTA

PhDTA 19.1

b 74
NOTA

b 11.4
PyN5
b 2.4
12-pyN4A

b 0.02
nompa
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b 0.16
dompa

b 2.4
pcma
a 0.02
NJC-L1

a 2.8
NJC-L2

a 55
NJC-L3

a
Cu(II),
b
Zn(II).
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 Wahsner et al. Page 243

Table 10:

Summary of relaxivity, water exchange kinetics, electronic relaxation, thermodynamic stability (in 0.15M
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NMe4Cl electrolyte solution), and oxidation potentials of selected Eu(II) complexes.

r1 (mM−1s−1) q τm310 (ns) ΔH‡ T1e310 (ns) Log KML E1/2 (V)

Eu-2.2.2 a b 2 2 33.4, 30.6 c d f g h

2.1 , 2.7 1.1, 3.0 13.0 −0.21 , −0.336

Eu-2.2.2-Ph a b 2 10 39.9 c d -- h
3.7 , 3.3 1.2, 3.8 −.208

Eu-2.2.2-BiPh a b 2 12 31.1 c d -- --
4.2 , 4.8 1.3, 4.2

Eu-2.2.2-BiPh / b-CD a 2 12 -- c d -- --
8.7 39, 155

Eu-2.2.2-BiPh / poly-bCD a 2 12 -- c d -- --
16.6 56, 215

Eu-2.2.2-BiPh / HSA a 2 12 -- c d -- --
12.5 191, 763
Eu-2.2.2-Ph-F -- -- -- -- -- -- h
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−0.079

Eu-2.2.2-Me4 -- -- -- -- -- -- h
−0.169

Eu-2.2.2-Ph2 -- -- -- -- -- -- h
−0.211

Eu-2.2.2-S4-Ph -- -- -- -- -- -- h
−0.035

Eu-ODDM -- -- -- -- -- 13.1e −0.92

g

Eu-ODDA -- 1 -- 22.5 c
4.0, 15
d 9.9e −0.82
g

Eu-DTPA -- 1 -- 26.3 c
2.3, 8.7
d 10.1e −1.35
g

Eu-DOTAM-Gly4 -- -- -- -- -- -- g
−.701

a
1.4 T, 37 °C;
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b
7 T;
c
1.5 T,
d
3.0 T.
f
Determined indirectly via cyclic voltammetry methods
g
vs. Ag/AgCl,
h
vs. Fc/Fc+.
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 Wahsner et al. Page 244

Table 11:

Summary of T1-based activatable MRI contrast agents.

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Responsive MRI probe Stimulus Mechanistic change r1 switch (mM−1s−1) % change Ref.

Gd-DOTA-4AmP pH Second sphere 3.5 to 5.3 (pH 9.5 to 51% increase 574–575
effect, τm 6) at 20 MHz

G5-PAMAM dendrimer pH Second sphere 10.8 to 24.0 (pH 9.5 122% increase 576
effect, τm to 6) at 20 MHz

Gd-DOTA-tetramide-hydroxypyridyl pH Second sphere effect ~5.6 to ~3.1 (pH 8.5 85% decrease 577
to 6) at 20 MHz

Gd-DO3A-sulfonamide pH q ~5.4 to 8.0 (pH 7.4 48% increase 308

to 6.8) at 65.6 MHz

Gd-DO3A-p-nitrophenolic arm pH q 4.1 to 7.0 (pH 9 to 71% increase 580

5) at 20 MHz

Gd-aminoethyl-DO3A derivatives pH q (pH 9 to 5) at 20 average: ~100% increase 581–582

MHz
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Gd-DO3A-sulfonamide-Ga-AAZTA pH q 3.7 to 10.1 (pH 8.5 150% increase 589

to 5) at 20 MHz

Gd-DO3A-glucuronic acid β-glucuronidase q 4.99 to 3.63 (human 27% decrease 623

serum) at 60 MHz

Gd-DO3A-ethyl ester esterase q 5.7 to 10.5 at 20 84% increase 624

MHz

Gd-DTPA fatty acid complex lipase solubilty switch inactive form: - 625
insoluble active
form: 4.7 at 20 MHz
Gd-DOTA-PEG-peptide MMP solubility switch - - 626–628

Gd-loaded, protamine-linked liposomes trypsin solubilty switch 0.2 to 1.8 800 % increase 629

Gd-DTPA with a lysine-masked HSA TAFI τR 9.8 to 26.5 (37 °C) 150% increase 630
binding group at 20 MHz

Gd-DTPA-FPG β-galactosidase τR 7.6 to 15.5 (37 °C) 103% increase 631

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at 20 MHz

Gd-DTPA with masked HSA binding β-galactosidase τR 3.5 to 5.5 (37 °C) at 57% increase 632
group 20 MHz

Gd-DTPA-derivative linked to a peptide legumain τR 27.1 to 73.5 (in cell 170 % increase 633
lysate) at 20 MHz
Gd-D-DOTA peroxidase τR 3.75 to 11.5 at 20 200 % increase 635
MHz

C-SNAM caspase-3 τR 10.2 to 19.0 at 42.6 90 % increase 640

MHz

Mn-polyaminocarboxylates tyrosinase L1: release of - - 634

Mn(II) L3: τR

Gd-DO3A-merocyanine NADH q 5.6 to 8.6 at 60 MHz 55 % increase 670

Gd-4NO22MeOSA hypoxia q 2.1 to 3.6 at 20 MHz 70 % increase 672

Mn(II)-H2qtp1 hydrogen peroxide q 4.73 to 5.3 at 128 10 % increase 657

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MHz

Mn(II)-H4qtp2 hydrogen peroxide q 5.46 to 7.17 at 128 30 % increase 675

MHz

Gd-DO3AS-Act free thiols q 8.1 to 4.1 at 20 MHz 50 % decrease 676

Gd-LC6-SH free thiols τR 5.3 to 2.33 at 200 55 % decrease 677–679

MHz

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 Wahsner et al. Page 245

Responsive MRI probe Stimulus Mechanistic change r1 switch (mM−1s−1) % change Ref.

Gd-SS-liposomes free thiols/free radicals τR 13.6 to 6.5 s−1 mM−1 50 % decrease 681
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at 20 MHz

Supramolecular Mn-porphyrine adducts p(O2) oxidation 40.8 to 15.2 at 20 60 % decrease 382

MHz

Mn-HBET glutathione/hydrogen peroxide reduction/oxidation Mn(II): 2.76 160 % increase/60 % 686

Mn(III): 1.05 at 60 decrease
MHz

Mn-JED L-cysteine/hydrogen peroxide reduction/oxidation Mn(II): 3.3 Mn(III): 560 % increase/80 % 689
0.5 at 60 MHz decrease

Gd-DTPA-bisamide derivative Zn(II) q 4.8 to 3.4 at 300 30 % decrease 696–697

MHz

Gd-DO3A-iminodiacetate Zn(II) q 2.3 – 5.1 at 60 MHz 120 % increase 698–699

Gd-CP027 Zn(II) τR 6.6 – 17.4 at 23 165 % increase 700

MHz

Mn-porphyrin-BPEN Zn(II) unknown 8.7 to 6.7 at 200 24% decrease 704

MHz
Author Manuscript

Gd-DOPTA Ca(II) q 3.26 – 5.76 at 500 80 % increase 706–707

MHz

Gd-BAPTA-ethyl esters esterase/Ca(II) q 7.6 to 12.6 at 60 66% increase 708

MHz

Gd-DOPTRA Ca(II) q 3.5 to 6.9 at 400 97% increase 709

MHz

Gd-DO3APBP Zn(II), Ca(II), Mg(II) τR - 200 – 500 % increase 710

Gd(III)-liposomes Ca(II) q, τR 7.3 to 38.1 at 20 400% increase 714

MHz

Gd-DO3A-thioether Cu(I) q 1.5 to 6.9 at 60 MHz 360% increase 715–716

Gd-DO3A-thioether-carboxylate Cu(I) q 2.6 to 11.4 at 60 340% increase 717

MHz

Gd-DO3A-thioether-octaarginine Cu(I) q 3.9 to 12.5 at 60 220% increase 718

MHz
Author Manuscript

Gd-QDOTAMA Cu(II) q 4.3 to 7.3 at 400 71% increase 719

MHz

Gd-DO2A-triazacrown ether zwitterionic neurotransmitter q at 300 MHz average: 70% decrease 728

Mn-porphyrin microgel temperature τR 8.4 to 14.5 at 128 73 % increase 729

MHz
Author Manuscript

Chem Rev. Author manuscript; available in PMC 2020 January 23.

 Wahsner et al. Page 246

Table 12:

Summary of activatable CEST agents.

Author Manuscript

Responsive MRI probe Stimulus Ref.

Dihydrouracil pH 8

Poly-L-lysine pH 591

Iopamidol pH 449

Iopromide pH 592

Iobitridol pH 590

L-arginine filled liposomes pH 593

Amine-based block copolymers pH 594

Imidazoles pH 595

Yb-DOTAM-Gly pH 600
Author Manuscript

Yb-DOTAM-poly(propylene imine) dendrimers pH 604

Tm-DOTAM-Gly-Lys pH 605

Eu-DOTAM-Gly-phenol pH 606, 609

Yb-DO3A-oAA pH 603

Yb-HPDO3A pH 610

Pyridine-Ln-DOTP-tButyl (ParaSHIFT) pH 613

Fe(II)-2-amino-6-picolyl-cyclen pH 612

DEVD-Tm-DOTA caspase-3 655

Cbz-GGR-Tm-DOTA urokinase plasminogen activator 657

Yb-DOTA-β-galactose β-galactosidase 659

Yb-DO3A-oAA-TML-ester esterase 661

Author Manuscript

Yb-DO3A-oAA-TML-Q DT-diaphorase 661

TM-DO3A-cadaverine transglutaminase 662

Cytosine cytosine deaminase 663

F5C cytosine deaminase 663

(LRRASLG)8 Protein Kinase A 664

(Phe-Arg)-4-amino-2-hydroxybenzoic acid Cathepsin B 665

Salicylic acid derivatives sulfatase or alkaline phosphatase 666–667

Salicylic acid derivatives Simultaneous detection: esterase and sulfatase 666–668

Yb-DO3A-oAA NO 691

Eu-DOTAM-N-methylqunolinium NADH 692

Eu-DOTAM-anthryl 1O 201
Author Manuscript

Eu-DOTAM-nitroxide hypoxia 184

Gd(III) modified liposomes reducing environment 695

Eu(2.2.2)-liposomes oxidation 405

Triazamacrocyclic cobalt complex oxidation/reduction 693

Chem Rev. Author manuscript; available in PMC 2020 January 23.

 Wahsner et al. Page 247

Responsive MRI probe Stimulus Ref.

Eu-DOTAM-BPEN Zn(II) 703
Author Manuscript

Yb-DOTA-tetraamide-iminodiacetate Ca(II)/Mg(II) 711

polymeric Eu-DOTAM derivative DNA 737

Author Manuscript
Author Manuscript
Author Manuscript

Chem Rev. Author manuscript; available in PMC 2020 January 23.