19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022].

See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Received: 14 August 2019 Revised: 4 January 2020 Accepted: 4 February 2020
DOI: 10.1002/wnan.1622

ADVANCED REVIEW

Molecular imaging of extracellular matrix proteins with

targeted probes using magnetic resonance imaging

Mani Salarian1 | Oluwatosin Y. Ibhagui1 | Jenny J. Yang1,2

1
Department of Chemistry, Georgia State
University, Atlanta, Georgia
Abstract
2
Center for Diagnostics and Therapeutics, The extracellular matrix (ECM) consists of proteins and carbohydrates that
Georgia State University, Atlanta, Georgia supports different biological structures and processes such as tissue develop-
ment, elasticity, and preservation of organ structure. Diseases involving
Correspondence
Jenny J. Yang, Department of Chemistry, inflammation, fibrosis, tumor invasion, and injury are all attributed to the
Center for Diagnostics and Therapeutics, transition of the ECM from homeostasis to remodeling, which can significantly
Georgia State University, Atlanta, GA
30303.
change the biochemical and biomechanical features of ECM components.
Email: [email protected] While contrast agents have played an indispensable role in facilitating clinical
diagnosis of diseases using magnetic resonance imaging (MRI), there is a
Funding information
National Institute of Health (NIH), Grant/ strong need to develop novel biomarker-targeted imaging probes for in vivo
Award Numbers: R33 CA235319, visualization of biological processes and pathological alterations at a cellular
R42AA112713, R42CA183376
and molecular level, for both early diagnosis and monitoring drug treatment.
Herein, we will first review the pathological accumulation and characteriza-
tion of ECM proteins recognized as important molecular features of diseases.
Developments in MRI probes targeting ECM proteins such as collagen, fibro-
nectin, and elastin via conjugation of existing contrast agents to targeting moi-
eties and their applications to various diseases, are also reviewed. We have
also reviewed our progress in the development of collagen-targeted protein
MRI contrast agent with significant improvement in relaxivity and metal bind-
ing specificity, and their applications in early detection of fibrosis and meta-
static cancer.

This article is categorized under:

Diagnostic Tools > in vivo Nanodiagnostics and Imaging
Biology-Inspired Nanomaterials > Peptide-Based Structures
Biology-Inspired Nanomaterials > Protein and Virus-Based Structures

KEYWORDS
cancer, extracellular matrix, fibrosis, magnetic resonance imaging, targeted probe

1 | INTRODUCTION

Mortality rates associated with cancer are due primarily to invasion and metastasis, which are fundamental features of
tumor biology. In industrialized countries, approximately 45% of deaths are caused by organ fibrosis (Fang, Yuan,
Peng, & Li, 2014). Cardiovascular diseases have the highest mortality rate in the United States in which nearly 50% of
these deaths are due to acute myocardial infarction (Krishnan et al., 2017). The most common feature in many of these

WIREs Nanomed Nanobiotechnol. 2020;12:e1622. wires.wiley.com/nanomed © 2020 Wiley Periodicals, Inc. 1 of 22

https://doi.org/10.1002/wnan.1622
 19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
2 of 22 SALARIAN ET AL.

diseases is remodeling of the extracellular matrix (ECM) in the microenvironment during disease development and pro-
gression. ECM remodeling is characterized by ECM proteins undergoing constant architectural changes, such as
degrading, redepositing, cross-linking, and stiffening (Fang et al., 2014), which then generates typical morphological
changes with significant impacts on the disease biology (Paszek et al., 2005). Many cardiovascular diseases, organ fibro-
sis and tumor biology, are all characterized by the accumulation of ECM components (Wynn, 2008). In case of organ
fibrosis, these mechanisms include aggravated injury-related responses and dysregulation during tissue repair processes
(Border & Noble, 1994; Friedman, Sheppard, Duffield, & Violette, 2013) involving increased production of a variety of
ECM components (Koyama & Brenner, 2017). As a result of abnormal ECM deposition, many diseases lead to disrup-
tion or distortion of tissue architecture and function (Murray, 2015) across a variety of organs, including connective tis-
sue, gastrointestinal tract, liver, kidney, lung, and heart (Friedman, 2015) (Figure 1).

1.1 | Role of ECM proteins in diseases

In each organ, the composition of the ECM has a distinct three-dimensional structure and pattern of constant remo-
deling for regulation of tissue homeostasis (Sonbol, 2018). Desmoplasia, involving stiffening, manipulation, induction,
and impairment of ECM, plays a role in many diseases, including chronic obstructive pulmonary disease, pancreatic
ductal adenocarcinoma, progression and metastasis of breast cancer, cardiomyopathy, inflammatory bowel disease,
chronic liver disease, atherosclerosis, and aortic aneurysm (Sonbol, 2018). Investigating the way in which the ECM reg-
ulates organ structure and function and how remodeling affects the development and progression of diseases may lead
to the improvement and discovery of new treatments; therefore, medical imaging with ECM-targeted probes has great
potential for a variety of diseases, with high clinical relevance (Box 1).

1.1.1 | Collagen

Collagen as the most significant and abundant component of the ECM in human tissue has 28 unique subtypes (Mouw,
Ou, & Weaver, 2014; Myllyharju & Kivirikko, 2004; Ricard-Blum, 2011; Ricard-Blum & Ruggiero, 2005). Each collagen
type is comprised of homotrimers or heterotrimers of left-handed helical α chains that are twisted to form a right-
handed triple helix structure (Ricard-Blum, 2011; Shoulders & Raines, 2009). Collagen includes a large superfamily of
proteins with Gly-X-Y motif, where X and Y are usually either proline or hydroxyproline (Bella, Eaton, Brodsky, &
Berman, 1994; Shoulders & Raines, 2009). Despite the large numbers of bulky proline residues in the structure, the heli-
ces are stabilized by glycine, interchain hydrogen bonds, and electrostatic interactions involving lysine and aspartate
(Walker, Mojares, & Del Rio Hernandez, 2018).
In lungs, collagen has two subtypes, type I and type VI collagen. The high accumulation of type VI collagen can
block airflow, causing chronic obstructive pulmonary disease (Bihlet et al., 2017; Kranenburg et al., 2006). In pancreatic
cancer (PC), in which 70% of patients die within the first year of diagnosis, collagens are the major components of the
tumor stroma of pancreatic ductal adenocarcinoma (PDAC). Types I, III, (Shields, Dangi-Garimella, Redig, & Munshi,
2012) and IV (Jablonska-Trypuc, Matejczyk, & Rosochacki, 2016) interstitial fibrillar collagen are vital for alterations in
PC-associated tissue homeostasis.
Major ECM protein collagen I is absent in normal tissue, or present at very low levels. In contrast, collagen I
desmoplastic stroma is characterized by up to a threefold increase in collagen compared with the normal pancreas
(Bailey et al., 2008; Lunardi, Muschel, & Brunner, 2014). Pathologically, PDAC has an intense fibrotic stromal response
or “desmoplastic reaction” which influences tumor survival and progression (Hidalgo, 2010). Evidence suggests that
cancer associated fibroblasts (CAF) or activated hepatic stellate cells (HSCs) produce the stromal collagen (Erkan,
2013a, 2013b; Haqq et al., 2014). Such stromal collagen expression is observed in patient matched PDAC and liver
metastasis. Importantly, collagen is overexpressed in the activated stroma subtype of PDAC and has been correlated
with poor clinical outcomes (Drifka et al., 2015; Li et al., 2018).
It has been shown that fibrillar collagen is reorganized and more aligned in PDAC patient samples in addition to its
remarkable abundance throughout the stroma. Periductal stroma collagen topology of PDAC differs from that of nor-
mal and chronic pancreatitis despite their upregulated collagen 1 expression levels. The stage-dependent collagen
expression density and spatial patterns of PDAC result from progressive crosslinking, degradation of collagen, and
stroma remodeling by lysyl oxidase (LOX) and matrix metalloproteinases (MMPs) during cancer progression. These
 19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SALARIAN ET AL. 3 of 22

F I G U R E 1 (a) Extracellular matrix components under normal conditions. (b) Extracellular matrix undergoes remodeling which is a
sequence of quantitative and qualitative alterations in its composition during disease development and progression. In fibrosis, cancer, and
many pathological conditions, this composition becomes dysregulated, stiff and more abundant

processes are frequently observed with other cancers, including breast cancer (Goodman, Rozypal, & Kelly, 2003)
(Figure 1), although the activity of LOX and MMPs usually reflects liver cirrhosis.
The spatial arrangement of collagen in stroma has also been observed in breast cancer and liver metastasis tissue
samples using second harmonic generation spectroscopy (Scodellaro et al., 2019; Wang et al., 2019). Collagen patterns
are associated with degree of tumor invasiveness (Pankova et al., 2016; Provenzano et al., 2008). Differential expression
levels of collagen type I and patterns are also reported to correlate to degree of liver fibrosis and its progression to later
stages, including liver cirrhosis, and likely contribute to its heterogeneity (Salarian, Turaga, et al., 2019).
Due to activation of stellate cells in the microenvironment in liver, collagen type I exhibits growth patterns similar
to many other types of cancers, including uveal melanoma, breast cancer, colon cancer, and PC (Figure 1)
 19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
4 of 22 SALARIAN ET AL.

BOX 1 FUNCTION AND IMPORTANCE OF EXTRACELLULAR MATRIX

Fibrotic diseases, such as pulmonary fibrosis, systemic sclerosis, liver cirrhosis, and cardiovascular disease, are
responsible for over 45% of deaths worldwide. Tumor metastasis accounts for 90% of cancer-related deaths.
These diseases represent the biggest challenges for healthcare systems due to lack of effective clinical treat-
ments. ECM remodeling is the common phenomenon in all these diseases. ECM composition and remodeling
are crucial for tumorigenesis and metastatic progression in cancer, as well as fibrotic disease.

(Grossniklaus, 2013; Yang & Grossniklaus, 2006). Grossniklaus et al. have shown that (Grossniklaus et al., 2016; Liao
et al., 2018) infiltrative pattern and portal vein patterns have very different arrangements of collagen alignment and
structural features, even at very early stages of liver metastasis (micrometastasis < 100 μm). It has been speculated that
these growth patterns could exhibit different responses to different therapies. Recently, we have shown that the growth
pattern of radiologically identified liver metastasis, determined by currently approved liver MRI contrast agents Eovist
or MultiHance, is largely inconsistent with pathologically defined patterns (Grossniklaus et al., 2016; Liao et al., 2018)
and can exhibit undetectable micrometastasis.
It is also important to note that stromal elements provide support for cancer cell survival and metastasis. In addi-
tion, dense collagen bundles at late disease stage mechanically block delivery of therapeutic agents to cancer cells
(Olive et al., 2009; Provenzano et al., 2012; Provenzano & Hingorani, 2013). Tumor permeability largely depends on
these spatial patterns of collagen with crosslinking. It is believed that the spatial alignment and expression level of
stroma collagen and low permeability in PDAC negatively correlate with survival. Evidence suggests that CAF or stel-
late cells and abnormal tumor blood vessels are two major contributors that lead to treatment failure. The choice of
treatment strategy is largely dependent on knowledge of stroma collagen expression (Dobner, Riechardt, Joussen,
Englert, & Bechrakis, 2012; Li, Yang, Chen, Alizadeh, & Niederkorn, 2009). Taken together, overexpression of collagen
is a good biomarker for diagnosis and staging of various diseases.

1.1.2 | Proteoglycans

Proteoglycans (PGs) are proteins characterized by covalent bonding with glycosaminoglycans (GAGs). These GAGs
consist of long chains of negatively charged disaccharide repeats in the form of heparin sulphate, chondroitin/dermatan
sulphate, hyaluronan (HA), or keratin sulphate. These GAGs can sequester water and cations because of their negative
charge, which give them space-filling and lubrication functions and properties (D'Ascenzo & Auffinger, 2015). In both
PDAC and breast cancer, the tumor stroma exhibit desmoplasia, which is rigid ECM containing PGs. In addition, the
GAG HA and PGs such as versican and aggrecan play roles in controlling the wound healing and fibrosis process
(Ghatak et al., 2015; Walker et al., 2018).

1.1.3 | Fibronectin and fibrin

Fibronectin (FN) is a multi-domain protein that interacts with different ECM components, therefore linking the cell to
the ECM (Mouw et al., 2014). Since there is alternative splicing of the mRNA, FN has 20 human isoforms encoded by a
single gene (Schwarzbauer & DeSimone, 2011; Singh, Carraher, & Schwarzbauer, 2010). FN forms a fibrillar network in
the ECM which is similar to collagen. FN is present as a dimer held together by two cysteine disulfide bonds outside
the cell, which is critical for stability and assembly in a fibrillar form (Schwarzbauer, 1991; Schwarzbauer & DeSimone,
2011). In various types of malignant tumors, FN is highly expressed and is associated with the tumor's invasiveness and
metastatic phenotype (Clark, Golub, Lander, & Hynes, 2000; Malik et al., 2010; Yao et al., 2007). It has been reported
that invasive ductal breast carcinoma and breast carcinoma demonstrate a six to sevenfold increase in FN expression
(Zhou et al., 2015). In addition, in liver fibrosis, FN is produced by HSCs, and has been shown to have an important role
in liver fibrogenesis, with excessive deposition occurring earlier than collagen deposition. In general, FN expression
 19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SALARIAN ET AL. 5 of 22

plays a critical role in mediating the long-term consequences of different chronic liver diseases (Leite et al., 2007; Liu
et al., 2016; Modol et al., 2015).

1.1.4 | Laminin

The basement membrane consists mostly of laminins which are a meshwork of proteins separating the epithelium,
mesothelium, and endothelium, from connective tissue. Laminin has 15 different subtypes, and each one has three sub-
chains (Sonbol, 2018). It has been reported that laminin and laminin fragments are important noninvasive fibrosis bio-
markers of alcoholic liver disease, viral hepatitis, and nonalcoholic fatty liver disease. It is also related to degrees of
fibrosis and severity of hepatitis and may reflect hepatic basement membrane metabolism (Mak, Chen, & Lee, 2013;
Mak & Mei, 2017).

1.1.5 | Elastin

The ECM elastin fibers are highly insoluble proteins that provide elasticity and resilience to tissues, allowing for their
reversible deformation. Elastin plays an important role in adult wound healing, and it is highly deficient and disorga-
nized during scar formation. It imparts recoil and resistance, and induces a range of cell activities, including cell migra-
tion and proliferation, matrix synthesis, and protease production (Perez-Rico et al., 2011, 2014). Elastin is highly
expressed in the media of the arterial vessel wall (Brasselet et al., 2005) and during plaque development, which there-
fore makes this ECM protein a promising biomarker for molecular imaging (Heeneman et al., 2003; Katsuda & Kaji,
2003). Furthermore, it is present during the initiation, progression, and complication of arterial diseases, including
abdominal aortic aneurysm (AAA) and atherosclerosis.

2 | ACCURATE D I S EAS E STAG I NG R E Q UI R E S NO NI NV A SI V E

MOLECULAR IMAGING

For optimal prognoses, it is imperative to accurately determine the stage of the disease, and to subsequently determine
the best therapeutic approach (Baues et al., 2017). For decades, needle-based biopsies were considered the gold standard
and were usually the only available option for specific and exact evaluation of disease differentiation (Feehally,
Floege, & Johnson, 2014; Fryer, Wang, Verrill, & Fleming, 2013; Popov & Schuppan, 2009). For cancer, biopsy remains
the current standard diagnostic procedure to assess the nature and stage of the tumor, and its malignancy
(Mahadevan & Von Hoff, 2007). Despite their disadvantages, needle-based biopsies still play an important role in the
diagnosis and staging of diseases. The main drawbacks include invasiveness with the risk of life-threatening complica-
tions, sampling errors, intraobserver and interobserver variability, and higher costs associated with patient hospitaliza-
tion (Myers, Fong, & Shaheen, 2008; Rousselet et al., 2005). Moreover, multiple biopsies are difficult and impractical,
due to the invasive nature of the procedure (Farrar et al., 2015). Due to the lack of noninvasive reliable tools and imag-
ing modalities to obtain information, usually up to four biopsies are still routinely taken in large phase III trials, for
example, in patients suffering from nonalcoholic steatohepatitis (NASH) (Rotman & Sanyal, 2017). In cancer, biopsy-
based diagnosis is particularly complicated, due to heterogeneity of the primary and metastatic tumors (Gerlinger et al.,
2012). Under these conditions, there is a high risk of spreading/seeding cancer cells, as well as increasing the accessibil-
ity of the pathological site (Robertson & Baxter, 2011). Liquid-based biopsies hold great promise for cancer diagnosis
and fibrotic diseases because of their high applicability, reproducibility and widespread availability. However, they may
not be organ-specific, and show limited indication of impaired organ function or inflammatory states. In addition, they
cannot accurately distinguish between different stages of disease (European Association for Study of Liver, Asociacion
Latinoamericana para el Estudio del Higado, 2015).
Noninvasive imaging modalities such as computed tomography (CT), magnetic resonance imaging (MRI), ultra-
sound, positron emission tomography (PET), and second near-infrared (Ding et al., 2019; Sun, Ding, Chen, et al., 2019;
Sun, Ding, Zhou, et al., 2019; Zhang et al., 2019) allow for the assessment of the diseased organ and provide anatomical
images mainly focusing on the structure instead of the process, and imaging of tissues or cells outside of the living sys-
tems. However, there is an unmet medical need in accurate detection, characterization, and quantification of relevant
 19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
6 of 22 SALARIAN ET AL.

pathobiological processes involved in these diseases at molecular and cellular levels (Mankoff, 2007; Toczek &
Sadeghi, 2016).
The mandate of molecular imaging is to address diagnostic gaps in precision medicine that conventional tests have
failed to address adequately to improve patient care (Mankoff, 2007; Toczek & Sadeghi, 2016). Diagnostic gaps include
evaluation of atherosclerotic plaque vulnerability, early stage detection of liver fibrosis, improved assessment of liver
cirrhosis heterogeneity, identification of different tumor subtypes, differentiation of active versus benign and original
sites for metastatic tumors in breast and PC, and quantitative monitoring of disease progression and treatment effect,
especially for targeted therapy in high risk patients. By specifically targeting different biological properties of the tissue
and providing information on key pathophysiological processes, the application of molecular imaging in a clinical set-
ting can lead to early detection before any structural changes can be detected by an imaging modality; assessment of
severity and heterogeneity, and potential for progression over time; and evaluation of the effect of therapeutic interven-
tions to guide patient care and management.
The process of molecular imaging usually consists of administration of a targeted or labeled probe, detection of the
probe with an appropriate imaging modality, and generation of images that reflect the probe distribution, providing
information on biological properties of the imaged tissues. The nature of the probe can be different based on the imag-
ing modality and the target.
The most important components of ECM, including collagens, PGs, laminins, FN, and elastin, are appealing targets
for molecular imaging of diseases. In organ fibrosis and different cancers, ECM protein accumulation as well as their
concentration increases with disease progression. For example, the concentration of overexpressed collagen I is esti-
mated to be 1–20 μM, which is much higher than receptors expressed at the cell surface (Caravan et al., 2007). In addi-
tion, they are extracellular, which makes them accessible to probes. Targeted probes could therefore act as imaging
agents for these diseases, to improve efficacy of disease assessment (Caravan, 2009; Caravan et al., 2007). Furthermore,
a targeted probe providing a measure of ECM protein levels and patterns as well as their heterogenous distribution
would have utility in detection of early stages of a disease and replace biopsy. Molecular imaging techniques with these
targeted probes have the potential to improve not only imaging-based diagnosis and risk stratification, but also the
assessment of responses to therapy, enabling the detection of changes at a molecular/biological level before the onset of
morphological changes. Such an approach surpasses what is currently achievable with traditional imaging modalities.

3 | M OLECULAR IM AG I NG US ING M RI AND CONTRAS T AG ENTS

MRI is a nonionizing diagnostic imaging modality based on strong static magnetic fields with radiofrequency pulses to
generate high-resolution anatomical images of structures with a high soft-tissue contrast (Lin et al., 2015). Molecular
MRI involves the application of targeted probes which bind selectively to different molecular targets in specific cells or
tissues. Targeted molecular probes can then shorten the local longitudinal (T1) and/or transverse (T2/T2*) relaxation
times of free water protons and this effect can be observed by MRI (Botnar et al., 2015; Johnson et al., 1993). As a non-
invasive diagnostic technique, MRI has several advantages, such as high soft-tissue contrast, high spatial and temporal
resolution, and avoidance of any ionizing radiation and nephrotoxic iodide-based contrast media. Thus, MRI has a high
potential in molecular imaging for early detection and staging (Ammirati et al., 2014).
MRI contrast agent (CA) has been applied to more than 30% of clinical MRI scans to increase contrast of normal
and pathological tissues (Major & Meade, 2009). They have provided information on changes in the vascular volume,
perfusion, and permeability of the tissues/organs changes for >400 million clinical applications (Cuenod & Balvay,
2013). The majority of commercially available T1-based MRI contrast agents utilize Gd3+ complexes formed by small
chelators DTPA (diethylenetriamine pentaacetic acid) or DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid) derivatives (low molecular weight < 1 kDa) (Figure 2).
The paramagnetic effect of a contrast agent (or relaxivity, r1 or r2, represented per millimolar concentration of Gd3+,
in mM−1 s−1) is controlled by several key parameters which can be optimized when designing novel targeted probes.
These parameters are: the number of water molecules directly bound to the Gd3+ ion (q, hydration number); the water
exchange rate of water molecules with bulk water known as kex, and finally the most important factor is the rotational
correlation time of the complex, τR (Xue, Qiao, Pu, Cameron, & Yang, 2013).
Most clinically available contrast agents usually have one water molecule in the inner-sphere of the Gd3+ complexes
(Figure 2) with relaxivities between 3 and 5 mM−1 s−1 at 1.5 or 3 T (clinical magnetic fields). In addition, they distribute
throughout the body without a specific target (except for Vasovist). Some have shown organ specificity (liver) such as
 19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SALARIAN ET AL. 7 of 22

FIGURE 2 Chemical structures of current marketed and clinically approved Gd3+-based MRI contrast agents as of 2019 with their
chemical names, tradenames, generic names and FDA approval year. Eovist and MultiHance have been approved for hepatic and ECF
distribution

Eovist and MultiHance. The achievement of molecular imaging of ECM biomarkers, especially for overexpression of
ECM proteins at 1–20 μM for early detection of related diseases, requires the development of targeted probes with sig-
nificantly improved sensitivity (relaxivity) as well biomarker targeting capability. Strategies to develop ECM-targeted
MRI contrast agents include conjugation of a targeting moiety to existing single or multiple copies of contrast agents
and novel protein MRI contrast agents (Figure 3) (Box 2).

4 | ECM TAR G ETED CON T R AST A GE NT S B A SE D O N CO NJ UGA T I O N

4.1 | General strategy

One of the more commonly used methods of achieving target specificity is the direct conjugation of derivatives of clini-
cally approved contrast agents such as DTPA or ProHance to peptides, antibodies, or small organic molecules
(Figure 3). The contrast agent usually consists of three parts: a targeting moiety, a unit which causes the contrast
enhancement, and a linker connecting the two parts. Either metalate-conjugate or conjugate-metalate techniques can
be used (Averill, Garcia, Siriwardena-Mahanama, Vithanarachchi, & Allen, 2011), to connect the Gd3+-complex or
contrast-enhancing unit to a targeting moiety. The conjugation process leads to the formation of amides, ethers,
thioesters, or triazoles, that are biologically stable (Vithanarachchi & Allen, 2012).
Monomeric targeted probes have one contrast-enhancing unit per targeting moiety. To overcome the limitation of
low relaxivity of existing contrast agents, multimeric contrast agents have been conjugated with one targeting moiety to
increase sensitivity to molecular biomarkers with low expression (Vithanarachchi & Allen, 2012). Other strategies
include the development of macromolecules (mainly nanoparticles) that incorporate, within the same unit, a high pay-
load of Gd3+ ions and a targeting moiety (Sanders et al., 2009; Vithanarachchi & Allen, 2012; Zhang et al., 2018). In
addition, T2-weighted contrast agents for cell tracking or specific targeting have also been developed (Ahrens & Bulte,
2013; Ding et al., 2017; Gu et al., 2018).
The affinity and specificity towards the target when designing a targeted probe is a major challenge. It is important
for a contrast agent to have proper specificity to ensure binding with the biomarker of interest. Most of the reported
targeted probes have affinities in the nM to μM range, which seems to be sufficient to perform an MRI. The length and
 19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
8 of 22 SALARIAN ET AL.

F I G U R E 3 Schematic illustration of different strategies for designing targeted contrast agents for extracellular matrix proteins. A
targeted MRI contrast agent consists of three main components: The Gd3+ chelate unit, linker, and targeting moiety (small molecule, peptide
or antibody)

BOX 2 PRECISION DIAGNOSTICS IN CHRONIC DISEASES WITH MOLECULAR IMAGING

The first step to achieve precision diagnostics in chronic diseases is to identify and validate biomarkers of early
disease which may likely different than those of late stage disease. The next step is to develop imaging agents
targeted to these new biomarkers. ECM proteins can be ideal disease biomarkers and concentration can
increase with disease progression to provide a quantitative level for disease stage (Early vs. Late). Their extracel-
lular location can make them readily accessible by the probe. Different patterns of the ECM proteins can reflect
the disease cause and stage. They provide information on the disease microenvironment as a reflection of the
mechanism, and finally, these ECM proteins are responsive to treatment to allow the monitoring of treatment
effects.

type of the linker can be different, based on the affinity towards the target. Moreover, it should not be affected by the
presence of the metal complex. It is also important to verify metal binding affinity and specificity of the ECM-targeted
contrast agents since these properties may be altered after conjugation.

4.2 | Molecular MRI of collagen using chelators

A type I collagen-specific peptide was identified using phage display and subsequently modified to improve its affinity
for collagen. The peptide consisted of 16 amino acids, GKWHCTTKFPHHYCLY, with three amino acids flanking a
cyclic peptide of 10 amino acids that is formed through a disulfide bond (Figure 4). It was concluded that
biphenylalanine at the amidated C terminus can improve collagen binding. Pioneered by Caravan et al., a collagen
targeted contrast agent EP-3533 was developed by conjugating three [Gd (DTPA)] moieties to the peptide targeting moi-
ety through thiourea linkages. EP-3533 has a collagen I binding affinity of 1.8 ± 1.0 μM (Caravan et al., 2007). This
 19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SALARIAN ET AL. 9 of 22

F I G U R E 4 (a) Chemical structures of developed collagen-targeted probes EP-3533 and CM-101 for molecular MR imaging of fibrosis.
(b) MR images of a control and fibrotic mouse and its correlation with histological characterization (collagen content), post injection of EP-
3533 in CCl4-induced liver fibrosis mouse model (Desogere, Montesi, & Caravan, 2019)

probe has a relaxivity of 16.1 mM−1 s−1 per Gd3+ (or 48.3 mM−1 s−1 per molecule) at 1.4 T. This contrast agent has been
applied to molecular imaging of collagen in multiple animal models.
In a mouse model of aged myocardial scar, EP-3533 has been shown to cause prolonged MR enhancement of the
collagen-rich scar, whereas the nonbinding isomer EP-3612 exhibited no such enhancement (Caravan et al., 2007; Helm
et al., 2008). Heart and blood samples were analyzed for Gd3+ content, indicating that there was more than twice as
much Gd3+ in the infarct zone, compared to remote myocardium 1 hr post-injection, and five times more Gd3+ in the
infarct than in the blood. In addition to the myocardial fibrosis model, Caravan et al. reported that a related probe could
be applied to target normal myocardium for a steady-state myocardial perfusion application (Spuentrup et al., 2009).
EP-3533 was also shown to be effective for detecting liver fibrosis (Polasek et al., 2012). In the study, a liver fibrosis
model induced by CCl4 over a period of weeks was used (Fuchs et al., 2013). It was shown that the MR signal change,
which was expressed as the change in contrast between liver and muscle, linearly correlated with hydroxyproline, dem-
onstrating total collagen concentration. The results were further confirmed with histology using Sirius Red staining and
quantification. It was concluded that the collagen-targeted probe could quantitatively stage fibrosis in multiple animal
models of liver fibrosis (Fuchs et al., 2013; Polasek et al., 2012) and assess response to antifibrotic therapies with high
LOX and LOXLn enzymes. These enzymes are upregulated (Jeay, Pianetti, Kagan, & Sonenshein, 2003; Li et al., 2003)
and oxidize the ε-amino groups of certain lysine and hydroxylysine residues to the aldehyde allysine concentration. In
another model of lung fibrosis induced by bleomycin, EP-3533 was shown to be effective by generating MRI signal
enhancement and therefore could detect fibrosis. Detection of pulmonary fibrosis in this model was further shown by
the probe uptake and MRI signal correlation with the amount of fibrosis determined by hydroxyproline (Fuchs et al.,
2013). In a more recent study, a new molecular MRI probe CM-101 was developed by using the same collagen-targeting
mechanism, but employing another macrocyclic chelate, Gd-DOTA, to facilitate clinical translation (Farrar et al., 2018).
This probe was utilized to detect liver fibrosis in two animal models as an alternative to linear gadolinium contrast
agents, which have been associated with development of nephrogenic systemic fibrosis (NSF) in patients (Sanyal,
Marckmann, Scherer, & Abraham, 2011; Thakral & Abraham, 2009).
In another study, an MRI contrast agent was developed based on high density lipoprotein (HDL) nanoparticles to
noninvasively visualize intraplaque collagen content in mouse atherosclerotic plaques (Chen et al., 2013). To generate
nanoparticles for collagen imaging, Chen et al. used labeled HDL nanoparticles and functionalized them with EP-3533
peptides (EP-3533-HDL) via poly(ethylene glycol) (PEG) spacers embedded in the HDL lipid layers. They reported a
relaxivity r1 of 9 ± 1 s−1 mM−1 (Chen et al., 2013).
 19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
10 of 22 SALARIAN ET AL.

Microbial surface component recognizing adhesive matrix molecules (MSCRAMM) represents a family of collagen bind-
ing adhesins with structural similarity different from conventional collagen I binding domains in integrin. Zong et al.
designed a collagen targeting molecule CNA-35, based on MSCRAMM type found in Staphylococcus aureus. CNA-35 pos-
sesses a relaxivity value per Gd3+ of 14 s−1 mM−1 (Klink et al., 2011), and a collagen binding affinity of 0.2 ± 0.02 μM. CNA-
35 was employed in the MRI of atherosclerosis plaques. CNA-35 together with high-resolution, multisequence MRI enabled
the monitoring of AAA in TGF-β neutralizing combined with angiotensin-induced AAA mice model (Zong et al., 2005).

4.3 | Molecular MRI of elastin and protoelastin

Makowski et al. developed an elastin-specific MRI contrast agent called “ESMA” for noninvasive quantification of
plaque burden in a mouse model of atherosclerosis (Figure 5). The MR signal generated by ESMA allowed for imaging
and evaluation of plaque burden. Furthermore, intraplaque elastin content was quantified by signal intensity to charac-
terize the plaque. The presence of high elastin content as well as its changes during plaque development, combined
with ESMA, demonstrated the potential of the contrast agent for noninvasive imaging of plaque burden with MRI
(Makowski et al., 2011). The contrast agent was first designed based on a DTPA structure, and then further developed
as a DOTA derivative and conjugated to a single amino acid D-phenylalanine. The relaxivity of Gd-ESMA was reported
as 4.5 mM−1 s−1, and when bound to elastin, increased to 13.7 mM−1 s−1 at 1.5 T. The contrast agent had a binding
affinity (Kd) of 1 ± 0.5 μM for elastin and 9.2 ± 0.7 μM for tropoelastin, the soluble monomer of elastin (Botnar et al.,
2014; Protti et al., 2015). Elastin levels were quantified at different stages of AAA by the contrast agent. In addition, Gd-
ESMA exhibited the potential for noninvasive detection of the aortic rupture site prior to dilation of the aorta and the
subsequent in vivo monitoring of compensatory repair processes during the progression of AAAs (Botnar et al., 2014).
In another study, ESMA accurately measured elastin bound gadolinium within the aortic wall and detected a decrease
in aortic wall elastin in mouse model with Marfan syndrome compared with wild-type controls. This study showed the
translational potential for noninvasively assessing aneurysm tissue changes and risk, as well as monitoring elastin con-
tent in response to therapeutic interventions (Okamura et al., 2014).
In order to improve the specificity of the contrast agent between elastin and tropoelastin, Phinikaridou et al. devel-
oped a probe targeting tropoelastin. This new contrast agent included a DOTA chelator in its structure, conjugated to a
15-amino acid peptide, VVGSPSAQDEASPLS. The new contrast agent, Gd-TESMA, provided selective targeting of tro-
poelastin versus the elastin (Phinikaridou et al., 2018). The targeting moiety peptide was chosen from an elastin-binding
protein (EBP) to tropoelastin. EBP is a subunit of the elastin/laminin receptor which is responsible for chaperoning the

F I G U R E 5 (a) Chemical structure of elastin-specific magnetic resonance contrast agent (ESMA). (b) DE-MR and time-of-flight (TOF)
angiogram images of brachiocephalic artery of a male ApoE−/− mouse with plaque burden in a model of atherosclerosis. Fusion of TOF and
high-resolution DE-MRI exhibits spatial registration of contrast uptake and luminal anatomy (Makowski et al., 2011)
 19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SALARIAN ET AL. 11 of 22

secreted tropoelastin to the extracellular space. The contrast agent had a binding affinity of 2.37 ± 0.05 μM for tropo-
elastin, and 14 ± 5 μM for elastin, which are similar to the range for Gd-ESMA. This new probe had a relaxivity of
11.7 ± 0.6 mM−1 s−1 and a relaxivity of 44 ± 1 mM−1 s−1 when bound to tropoelastin in vitro at 0.47 T. When used in a
murine model of atherosclerosis, the contrast agent demonstrated a lower in vivo relaxivity value of 7.15 mM−1 s−1 at
3 T. Recently, Sun, Baues, et al. (2019) reported the application of Gd-ESMA in molecular imaging of elastin, allowing
the noninvasive staging and longitudinal monitoring of renal fibrosis.

4.4 | Molecular MRI of FN and fibrin

Lu et al. reported the development of a peptide targeted contrast agent, CLT1-(Gd-DTPA), for MRI molecular imaging
of FN-fibrin complexes in mice bearing HT-29 human colon carcinoma xenografts (Ye et al., 2008). The targeted con-
trast agent is a conjugate of a cyclic decapeptide CGLIIQKNEC (CLT-1). They reported r1 and r2 relaxivities of 4.22 and
4.45 mM−1 s−1, respectively for CLT1-(Gd-DTPA) at 3 T (Ye et al., 2008). In another study, the same group reported a
pentapeptide CREKA (Cys-Arg-Glu-Lys-Ala)-targeted gadolinium-based MRI contrast agent, CREKA-Tris (Gd-DOTA)3
(Gd-DOTA (4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecyl gadolinium), which binds to fibrin and FN com-
plexes that are abundant in the tumor microenvironment of fast-growing breast cancers (Figure 6) (Zhou et al., 2015).
They assessed the capability of CREKA-Tris(Gd-DOTA)3 to detect micrometastasis with MRI in coregistration with
high-resolution fluorescence cryoimaging in female mice bearing metastatic 4T1 breast tumors. The r1 relaxivity of
CREKA-Tris-Gd (DOTA)3 was reported to be 8.06 mM−1 s−1 per Gd3+ (24.18 mM−1 s−1 per molecule) at room tempera-
ture and 3 T (Zhou et al., 2013).
A new MRI contrast agent targeting extradomain-B FN (EDB-FN), which is overexpressed in the ECM of aggressive
tumors, was also recently reported (Han et al., 2015; Li et al., 2017). This study identified four small peptides (GVK,
IGK, SGV, and ZD2) specific to EDB-FN, conjugating them to DOTA or HP-DO3A. The targeted probes were then
tested in the PC3 mouse prostate cancer model resulting in tumor contrast enhancement under MRI. The peptide, ZD2
(TVRTSAD), was conjugated to both HP-DO3A and DOTA chelates and the contrast agent, ZD2-(Gd(HP-DO3A))
exhibited a binding affinity of 1.7 μM with a relaxivity of 5.4 mM−1 s−1 at 1.5 T (Ayat et al., 2018).
Numerous studies have reported promising results, with potential translational applications, for the development of
fibrin-targeted MRI probes for detection of thrombus. Fibrin-targeted peptides, Tn7 (Nair et al., 2008; Zhang et al.,

F I G U R E 6 (a) Development of a pentapeptide CREKA (Cys-Arg-Glu-Lys-Ala)-targeted gadolinium-based MRI contrast agent, CREKA-
Tris(Gd-DOTA)3 binding to fibrin–fibronectin complexes and its application for detection of 4T1 breast tumor micrometastasis with MRI in
co-registration with high-resolution fluorescence cryo-imaging. (b) MR imaging of 4T1–GFP-Luc2 breast cancer cells using CREKA-Tris (Gd-
DOTA)3 (Zhou et al., 2015; Zhou, Wu, Kresak, Griswold, & Lu, 2013)
 19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
12 of 22 SALARIAN ET AL.

FIGURE 7 Development of fibrin-targeted Gd3+-based MRI contrast agent and its application in detection of thrombi in patients.
(a) Structure of the Gd3+-based fibrin-binding probe EP-2104R. (b) Molecular MR imaging of left ventricular (LV) thrombus. (c) MRI of
thrombus in the right atrium (arrow, clot surface; arrowhead, central venous catheter). Clot in the right atrium is shown in CT image.
(d) MRI of thrombus in the carotid artery and signal enhancement at the intraluminal clot surface post EP-2104R injection. (e) MRI of
thrombus in the descending thoracic aorta (arrow). CT image demonstrates plaque in the aortic wall. Arrowhead shows a small calcification
(Spuentrup et al., 2008)

2010) and Tn6 (Botnar et al., 2004; Overoye-Chan et al., 2008), were further modified with as many as four Gd3+ bind-
ing chelates to create an MRI probe with high relaxivity and stability. The Tn6-based contrast agent with four Gd-DTPA
moieties, EP-1873, demonstrated a relaxivity of 84 mM−1 s−1 at 1.4 T, 37 C per molecule and Kd of 3.5 μM for fibrin
(Botnar et al., 2004). A rabbit model of acute thrombosis after atherosclerotic plaque rupture was used to demonstrate
the applicability of EP-1873 for thrombus detection. The probe was further optimized to EP-2104R to improve fibrin
binding and stability with four Gd-DOTA moieties (Overoye-Chan et al., 2008) exhibiting a relaxivity of 71.4 mM−1 s−1
per molecule at 1.4 T and 37 C. The contrast agent, EP-2104R was applied in rat, rabbit, and pig models with arterial
thrombus (Botnar et al., 2004), venous thrombus (Stracke et al., 2007), pulmonary and cerebral embolism (Spuentrup,
Katoh, et al., 2005; Uppal et al., 2010), coronary thrombosis (Spuentrup, Buecker, et al., 2005), and atrial thrombus
(Spuentrup, Fausten, et al., 2005). It was also shown that EP-2104R was able to identify thrombi that are prone to
thrombolysis by estimating thrombus composition related to fibrin content (Andia et al., 2014). Application of EP-
2104R in humans was further investigated in two phase II trials with 52 patients which had confirmed thrombi in the
venous, heart or arterial systems (Spuentrup et al., 2008; Vymazal et al., 2009). With a clinical 1.5 T scanner, EP-2104R
was shown to be able to detect thrombi in the left ventricle, right atrium, carotid artery and thoracic aorta with molecu-
lar imaging of fibrin in humans (Figure 7) (Oliveira & Caravan, 2017; Spuentrup et al., 2008).
With promising results from the MR probe, EP-2104R, in detection of thrombi in humans, Caravan et al. further opti-
mized a bimodal PET/MRI probe designated 64Cu-EP-2104R. Using this contrast agent, MR image demonstrated the true
size of the thrombus while the PET images showed an overestimate of the true thrombus size (Uppal et al., 2011).
Aime et al. introduced an AAZTA-tetramer conjugated to the same fibrin-binding peptide used for EP-2104R
(XnCPYGLCXm) developed by Caravan et al. (Overoye-Chan et al., 2008; Tripepi et al., 2018). This newly designed,
targeted probe, FibPep-(Gd-AAZTA)4, incorporated four Gd (III)-AAZTA chelate functions, which were coupled to a
trilysine scaffold, functionalized with a maleimide function, then conjugated to the cyclic 11-amino acid fibrin-binding
peptide (FibPep) (Overoye-Chan et al., 2008). The targeted probe had a relaxivity of 18.5 ± 0.3 mM−1 s−1 per Gd3+ or
74 ± 1 mM−1 s−1 per molecule in water. It had a higher relaxivity of 58 ± 13 mM−1 s−1 per Gd3+ in the presence of
fibrin plasma clots at 0.5 T, and a binding affinity (Kd) of 3.0 ± 0.6 μM was reported (Tripepi et al., 2018). A summary of
main contrast agents for molecular imaging of ECM proteins is shown in Table 1.

5 | D E S I G N OF TA R G E T E D C O N T R AS T A G E N T S U S I N G P R O T E I N S

Conceptually different from using existing MRI contrast agents based on small chelators, we have created a novel plat-
form for developing protein MRI contrast agents (ProCAs) (Figures 3 and 7). This novel platform technology allows us
 TABLE 1 Molecular imaging agents for imaging of ECM proteins
SALARIAN ET AL.

Contrast Kd Target Relaxivity (r1) per Gd3+

agent (μM) Source protein Disease model (mM−1 s−1) at 1.5 T References
ProCA32. 1.42 Protein Collagen Fibrosis (liver, lung) pancreatic cancer, 34.00 Cave, Daures, Parello, Saint-Yves,
collagen1 engineering type 1 liver metastasis, COPD, aortic aneurysm and Sempere (1979), Salarian,
Turaga, et al. (2019)
EP-3533 1.80 Phage display Collagen Fibrosis (liver, lung, muscular) pancreatic cancer 16.10 Caravan et al. (2007)
type 1 atherosclerosis
CM-101 3.60 Phage display Collagen Liver fibrosis 11.10 Farrar et al. (2018)
type 1
CNA-35 0.20 Natural protein Collagen Aortic aneurysm 14.00 Klink et al. (2011)
sequence type 1
ESMA 1.0 Chemical synthesis Elastin Aortic aneurysm, fibrosis (kidney, 4.50 Makowski et al. (2011)
liver) myocardial infarction,
atherosclerosis
ESMA 9.20 Chemical synthesis Tropoelastin N/A 13.70 Botnar et al. (2014), Protti et al. (2015)
a
Gd-TESMA 2.37 Chemical synthesis Tropoelastin Atherosclerosis 44.00 Phinikaridou et al. (2018)
b
CLT-1 N/A Phage display Fibronectin- Colon cancer 4.22 Ye et al. (2008)
fibril
CREKA N/A Phage display Fibronectin- Breast cancer micro metastasis 8.06b Zhou et al. (2015)
fibril
AAZTA N/A SPPSc Fibril N/A 7.10a Uppal et al. (2011)
d
EP-2104R 1.7 SPPS Fibril Thrombi 17.4 Botnar et al. (2004)
FibPep 3.00 Phage display Fibril Thrombosis 18.50 Botnar et al. (2004)
ZD2 1.70 Phage display Fibronectin Prostate cancer 5.40 Ayat et al. (2018)

Abbreviation: ECM, extracellular matrix.

a
Relaxivity values measured at 0.47 T.
b
Relaxivity values measured at 3 T.
c
SPPS: solid phase peptide synthesis.
d
SPPS: a combination of solid and solution phase peptide synthesis.
13 of 22

19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
 19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
14 of 22 SALARIAN ET AL.

to systematically optimize relaxivity, metal binding affinity and selectivity, targeting capability, and in vivo properties
(Li et al., 2012; Pu et al., 2015; Pu, Salarian, et al., 2016; Qiao et al., 2014; Xue et al., 2013, 2014, 2015). These proteins
use side chains from the scaffold protein to directly generate a Gd3+ binding protein and the designed protein itself
serves as a chelator to tightly bind to Gd3+. In this approach, MRI contrast agents with high relaxivity can be achieved
by improving the three key factors of τR, q, and outer sphere water contributions, without sacrificing desired in vivo dif-
fusion of water and stability of Gd3+. In addition, we have also created a new generation of ProCA agent with two Gd3+
binding sites (ProCA32) by reshaping two Ca2+ binding sites in parvalbumin and surface PEGylation (ProCA32-P40
stands for the PEGylated version of ProCA32) (Xue et al., 2015). The Gd3+ binding constant for ProCA32 is significantly
improved and is comparable to DTPA. Strikingly, ProCA32 exhibits a Gd3+ dissociation constant comparable to that of
DTPA of 1.86 × 10−21 M, while wild type parvalbumin has a Gd3+ affinity of 5 × 10−12 M (Cave et al., 1979). ProCA32
achieves the greatest metal selectivity for Gd3+ over Ca2+, Mg2+, Zn2+ and other physiological metal ions that are >100
to 1011-fold higher than DTPA.
Biomarker targeted protein contrast agents have been further developed by engineering a targeting moiety to pro-
tein contrast agents such as ProCA1 and ProCA32 with a flexible hinge or linker to provide the flexibility required for a
targeting moiety (Li et al., 2012; Pu et al., 2015; Pu, Salarian, et al., 2016; Pu, Xue, Qiao, Patel, & Yang, 2016; Xue et al.,
2013, 2014, 2015; Yang et al., 2008). This approach enables us to add molecular specificity, without sacrificing metal
binding and relaxivity properties (Pu et al., 2015; Qiao et al., 2011; Wei et al., 2011). In addition, we have developed a
collagen targeted protein contrast agent, ProCA32.collagen1 (Salarian, Turaga, et al., 2019; Salarian, Yang, et al., 2019)
by engineering a collagen type I targeting moiety to the C-terminal of protein contrast agent, ProCA32 (with two Gd3+
binding sites). In the modeled structure, lysine residues are positioned as anchor points for PEG modification. A flexible
hinge was used to maximize targeting capacity and relaxivity, while maintaining metal binding capability (Salarian,
Turaga, et al., 2019; Salarian, Yang, et al., 2019). PEGylation was used to improve relaxivities by tuning correlation time.
It also increases stability and blood retention time (Li et al., 2012). ProCA32.collagen1 possesses high relaxivities per
particle (r1 and r2) at both 1.4 and 7.0 T, which enables robust detection of early-stage (Ishak stage 3 of 6) liver fibrosis
and NASH (Ishak stage 1 of 6 or 1A mild) in animal models via dual contrast modes. ProCA32.collagen1 also identifies
vasculature changes associated with intrahepatic angiogenesis and portal hypertension during late-stage fibrosis, and
heterogeneity (Figure 9) via serial molecular imaging. This developed ProCA32.collagen1 also enables the detection of
micrometastasis and largely improves the current detection limit from ~10–20 mm to 0.144 mm in mouse models
(Figures 8 and 9). We further demonstrated that ProCA32.collagen1 is likely to mitigate metal toxicity due to lower dos-
age and strong resistance to transmetallation as well as unprecedented metal selectivity that is 102 and 1013-fold higher
for Gd3+ over Ca2+ and Zn2+, respectively, than clinical “safe” contrast agents.

6 | B A R R I E R S I N C L I N I C A L TR A N S L A T I O N O F M R I CO N T R A S T A G E N T S

Before 1994, only three MRI contrast agents had been approved for clinical use in the United States. As of 2019, four
more contrast agents had been approved by the Food and Drug Administration (FDA) and are still prescribed and
marketed across the United States (Figure 2). In 2017, two FDA approved contrast agents, Gadofosveset trisodium
(Ablavar) and Gadoversetamide (Optimark) were discontinued by their manufacturers due to poor sales or similarities
to other agents (Fotenos, 2018; Ibrahim, Hazhirkarzar, Gupta & Dublin, 2020). Over the past 30 years, many promising
biomarker targeted Gd3+-based molecular imaging agents have been developed and successfully demonstrated in proof-
of-concept animal studies, but apart from those seven nontargeted contrast agents, none has obtained clinical approval.
Despite the tremendous effort and progress made in developing new types of MRI contrast agents with or without
targeting capability, translation into clinical practice has encountered several major challenges. One of the biggest bar-
riers has been the safety related to metal toxicity. In the past 30 years of development of Gd3+ MRI contrast agents, con-
cerns have been reported regarding potential NSF effects associated with the approved contrast agents, especially linear
ones such as Omniscan with weaker Gd3+ binding affinity. In 2017, reports of Gd3+ deposition in the brain raised addi-
tional concerns and limited or suspended the applications of intravenous linear contrast agents, such as Omniscan and
Magnevist, in the European Union as recommended by European Medicines Agency (European Medicines Agency,
2017). Eovist and Multihance were exempted or restricted to only liver applications due to lack of alternatives. It is
worth mentioning that the majority of small Gd3+-binding chelators have weak metal selectivity for Gd3+ over physio-
logical metal ions such as Ca2+, Zn2+, and Mg2+. Our developed protein-based MRI contrast agents, with higher metal
selectivity, exhibit strong advantages over these small chelators (Pu et al., 2015; Pu, Salarian, et al., 2016; Salarian,
 19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SALARIAN ET AL. 15 of 22

F I G U R E 8 Design and development of collagen I targeted protein-based MRI contrast agent (ProCA32.collagen1) for detection of
chronic liver diseases (liver fibrosis and metastasis). ProCA32.collagen1 was developed by covalently linking a collagen type I targeting
peptide moiety consisting of natural amino acids to the C-terminal of protein contrast agent ProCA32 (with two Gd3+ binding sites).
PEGylation of the contrast agent was performed to improve protein solubility, blood retention time, and reduce immunogenicity (Salarian,
Turaga, et al., 2019; Salarian, Yang, et al., 2019)

F I G U R E 9 (a) Molecular MR imaging of collagen heterogeneity in liver cirrhosis (three models of NASH, TAA/alcohol, and
diethylnitrosamine) with ProCA32.collagen1. (b) Detection of collagen heterogeneity in uveal melanoma liver metastasis using ProCA32.
collagen1. Different collagen patterns and distribution in the liver and tumor, and its correlation with histology, can be observed in all four
animal models
 19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
16 of 22 SALARIAN ET AL.

Turaga, et al., 2019; Salarian, Yang, et al., 2019; Xue et al., 2015). Moreover, it is essential to ensure that metal binding
affinity and relaxivity of targeted contrast agents are not reduced upon addition of a targeting moiety. Unfortunately,
while many ECM targeted contrast agents have been developed by conjugation of the targeting moiety to the existing
approved contrast agents, their metal binding affinity and selectivity properties are often not reported. Determination
of metal selectivity constants is imperative since it is known that small molecules are sensitive to any structural modifi-
cations (Wadas, Wong, Weisman, & Anderson, 2010).
Another challenge is that toxicology tests are costly and time consuming and require extensive funding that can
often be difficult to obtain. In addition, the revenue generated by clinically approved molecular imaging agents is much
lower than that of the clinically approved pharmaceuticals, partly because of the single or infrequent use of these imag-
ing probes compared to daily or frequent drug use. In order to address this challenge, a targeted molecular imaging
probe can be linked to a pharmaceutical drug so that the imaging diagnosis can contribute to the therapy to enhance
personalized medicine.
Furthermore, molecular imaging for low-expression molecular biomarkers requires significantly improved relaxivity
and specificity to overcome in vivo background noise. Targeted contrast agents require strong tumor and tissue penetra-
tion. The large size of both antibodies and nanoparticles results in limited tissue penetration capability and very slow
in vivo targeting. In addition, the development of new image processing techniques can ensure more accurate and pre-
cise correlation of radiology and histology data for both small animal and subsequent patient translational applications.
Targeted MRI contrast agents are often developed to facilitate imaging by providing a ratio of MR signals that corre-
sponds to the biomarker with adequate sensitivity, without being affected by background signal in tissues. Therefore, it
is imperative that the ratio be independent of the concentration of the probe in tissue, in order to avoid major complica-
tions related to interpreting molecular imaging results. However, validation in clinical trials of these newly developed
targeted probes requires addressing these many challenges.

7 | FUTURE DIRECTION

The main strategy to achieve an ECM-targeted contrast agent depends on several factors. The two main components
are the Gd3+ chelate unit, responsible for generating MRI signal, and the targeting moiety, responsible for the recogni-
tion of the desired ECM protein. There are many different conjugation strategies reported for development of targeted
probes; however, the translation of these contrast agents into the clinic is a challenging process. The developed bio-
marker targeted contrast agents need to exhibit strong thermodynamic stability and kinetic inertness of the Gd3+ che-
lating unit upon addition of a targeting moiety, with strong affinity and specificity towards the desired target. They
should exhibit improved relaxivity required for the detection of low concentration of ECM biomarkers while demon-
strating superior pharmacokinetic properties, and fast clearance. In addition, the targeted probe requires good acquisi-
tion time or imaging window with no in vivo toxicity. A better understanding of the structure of the ligands, along with
choice of a good target, facilitates the rational design of targeted probes. Recently, there has been an interest in develop-
ment of multimodal imaging probes; however, the clinical application for these probes has yet to be determined. Choos-
ing the appropriate diagnostic biomarkers to gain the most benefit from the technology and the difference in sensitivity
between the modalities have been a great challenge. The current nontargeted multimodal probes such as 64Cu-labeled
magnetic nanoparticles (Ai, Ferreira, Chen, & Cai, 2016; Glaus, Rossin, Welch, & Bao, 2010) lack the required sensitiv-
ity. This probe can be a good example to demonstrate that development of a PET/MRI or SPECT/MRI imaging agent
cannot necessarily compensate for the large differences in sensitivities between the two modalities. Therefore, multi-
modal probes optimized for both will require large amounts of low-sensitivity probes (MRI) that will increase probe size
compared with the single high sensitivity probe (PET or SPECT). However, more recently, development of fibrin-
targeted PET/MRI probes such as 64Cu-EP-2104R (Uppal et al., 2011) have shown very promising results with human
translational potential for molecular imaging of ECM proteins.
Tremendous effort will be required to overcome these challenges in moving from the translational stage into the
clinic. Further development of ECM targeted MRI contrast agents will also benefit from addressing specificity, heteroge-
neity, and various patterns of targets, in both biological and pathological states through detailed correlation of key fea-
tures of animal imaging to pathological analysis via machine learning and artificial intelligence.

A C K N O WL E D G M E N T
We thank Dr Michael Kirberger for carefully editing the manuscript.
 19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SALARIAN ET AL. 17 of 22

CONFLICT OF INTEREST
The authors have declared no conflicts of interest for this article.

A U T H O R C ON T R I B U T I O NS
Mani Salarian: Writing-original draft; writing-review and editing. Oluwatosin Ibhagui: Writing-review and editing.
Jenny Yang: Funding acquisition; supervision; writing-review and editing.

ORCID
Jenny J. Yang https://orcid.org/0000-0002-2310-7658

R EL A TE D WIR ES AR TI CLE
Design of a novel class of protein-based magnetic resonance imaging contrast agents for the molecular imaging of
cancer biomarkers

R EF E RE N C E S
Ahrens, E. T., & Bulte, J. W. (2013). Tracking immune cells in vivo using magnetic resonance imaging. Nature Reviews. Immunology, 13,
755–763.
Ai, F., Ferreira, C. A., Chen, F., & Cai, W. (2016). Engineering of radiolabeled iron oxide nanoparticles for dual-modality imaging. WIREs
Nanomedicine and Nanobiotechnology, 8, 619–630.
Ammirati, E., Moroni, F., Pedrotti, P., Scotti, I., Magnoni, M., Bozzolo, E. P., … Camici, P. G. (2014). Non-invasive imaging of vascular
inflammation. Frontiers in Immunology, 5, 399.
Andia, M. E., Saha, P., Jenkins, J., Modarai, B., Wiethoff, A. J., Phinikaridou, A., … Botnar, R. M. (2014). Fibrin-targeted magnetic resonance
imaging allows in vivo quantification of thrombus fibrin content and identifies thrombi amenable for thrombolysis. Arteriosclerosis,
Thrombosis, and Vascular Biology, 34, 1193–1198.
Averill, D. J., Garcia, J., Siriwardena-Mahanama, B. N., Vithanarachchi, S. M., & Allen, M. J. (2011). Preparation, purification, and character-
ization of lanthanide complexes for use as contrast agents for magnetic resonance imaging. Journal of Visualized Experiments.
Ayat, N. R., Qin, J. C., Cheng, H., Roelle, S., Gao, S., Li, Y., & Lu, Z. R. (2018). Optimization of ZD2 peptide targeted Gd(HP-DO3A) for detec-
tion and risk-stratification of prostate cancer with MRI. ACS Medicinal Chemistry Letters, 9, 730–735.
Bailey, J. M., Swanson, B. J., Hamada, T., Eggers, J. P., Singh, P. K., Caffery, T., … Hollingsworth, M. A. (2008). Sonic hedgehog promotes
desmoplasia in pancreatic cancer. Clinical Cancer Research, 14, 5995–6004.
Baues, M., Dasgupta, A., Ehling, J., Prakash, J., Boor, P., Tacke, F., … Lammers, T. (2017). Fibrosis imaging: Current concepts and future
directions. Advanced Drug Delivery Reviews, 121, 9–26.
Bella, J., Eaton, M., Brodsky, B., & Berman, H. M. (1994). Crystal and molecular structure of a collagen-like peptide at 1.9 A resolution. Sci-
ence, 266, 75–81.
Bihlet, A. R., Karsdal, M. A., Sand, J. M., Leeming, D. J., Roberts, M., White, W., & Bowler, R. (2017). Biomarkers of extracellular matrix
turnover are associated with emphysema and eosinophilic-bronchitis in COPD. Respiratory Research, 18, 22.
Border, W. A., & Noble, N. A. (1994). Transforming growth factor beta in tissue fibrosis. The New England Journal of Medicine, 331,
1286–1292.
Botnar, R. M., Ebersberger, H., Noerenberg, D., Jansen, C. H., Wiethoff, A. J., Schuster, A., … Makowski, M. R. (2015). Molecular imaging in
cardiovascular diseases. Rofo, 36, 92–101.
Botnar, R. M., Perez, A. S., Witte, S., Wiethoff, A. J., Laredo, J., Hamilton, J., … Johnstone, M. T. (2004). In vivo molecular imaging of acute
and subacute thrombosis using a fibrin-binding magnetic resonance imaging contrast agent. Circulation, 109, 2023–2029.
Botnar, R. M., Wiethoff, A. J., Ebersberger, U., Lacerda, S., Blume, U., Warley, A., … Makowski, M. R. (2014). In vivo assessment of aortic
aneurysm wall integrity using elastin-specific molecular magnetic resonance imaging. Circulation. Cardiovascular Imaging, 7, 679–689.
Brasselet, C., Durand, E., Addad, F., Al Haj Zen, A., Smeets, M. B., Laurent-Maquin, D., … Lafont, A. (2005). Collagen and elastin cross-
linking: A mechanism of constrictive remodeling after arterial injury. American Journal of Physiology. Heart and Circulatory Physiology,
289, H2228–H2233.
Caravan, P. (2009). Protein-targeted gadolinium-based magnetic resonance imaging (MRI) contrast agents: Design and mechanism of action.
Accounts of Chemical Research, 42, 851–862.
Caravan, P., Das, B., Dumas, S., Epstein, F. H., Helm, P. A., Jacques, V., … Zhang, Z. (2007). Collagen-targeted MRI contrast agent for molec-
ular imaging of fibrosis. Angewandte Chemie (International Ed. in English), 46, 8171–8173.
Cave, A., Daures, M. F., Parello, J., Saint-Yves, A., & Sempere, R. (1979). NMR studies of primary and secondary sites of parvalbumins using
the two paramagnetic probes Gd (III) and Mn (II). Biochimie, 61, 755–765.
Chen, W., Cormode, D. P., Vengrenyuk, Y., Herranz, B., Feig, J. E., Klink, A., … Fayad, Z. A. (2013). Collagen-specific peptide conjugated
HDL nanoparticles as MRI contrast agent to evaluate compositional changes in atherosclerotic plaque regression. JACC: Cardiovascular
Imaging, 6, 373–384.
 19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
18 of 22 SALARIAN ET AL.

Clark, E. A., Golub, T. R., Lander, E. S., & Hynes, R. O. (2000). Genomic analysis of metastasis reveals an essential role for RhoC. Nature,
406, 532–535.
Cuenod, C. A., & Balvay, D. (2013). Perfusion and vascular permeability: Basic concepts and measurement in DCE-CT and DCE-MRI. Diag-
nostic and Interventional Imaging, 94, 1187–1204.
D'Ascenzo, L., & Auffinger, P. (2015). A comprehensive classification and nomenclature of carboxyl-carboxyl(ate) supramolecular motifs and
related catemers: Implications for biomolecular systems. Acta Crystallographica. Section B: Structural Science, Crystal Engineering and
Materials, 71, 164–175.
Desogere, P., Montesi, S. B., & Caravan, P. (2019). Molecular probes for imaging fibrosis and fibrogenesis. Chemistry, 25, 1128–1141.
Ding, C., Wu, K., Wang, W., Guan, Z., Wang, L., Wang, X., … Fan, J. (2017). Synthesis of a cell penetrating peptide modified super-
paramagnetic iron oxide and MRI detection of bladder cancer. Oncotarget, 8, 4718–4729.
Ding, F., Chen, Z., Kim, W. Y., Sharma, A., Li, C., Ouyang, Q., … Kim, J. S. (2019). A nano-cocktail of an NIR-II emissive fluorophore and
organoplatinum(ii) metallacycle for efficient cancer imaging and therapy. Chemical Science, 10, 7023–7028.
Dobner, B. C., Riechardt, A. I., Joussen, A. M., Englert, S., & Bechrakis, N. E. (2012). Expression of haematogenous and lymphogenous che-
mokine receptors and their ligands on uveal melanoma in association with liver metastasis. Acta Ophthalmologica, 90, e638–e644.
Drifka, C. R., Tod, J., Loeffler, A. G., Liu, Y., Thomas, G. J., Eliceiri, K. W., & Kao, W. J. (2015). Periductal stromal collagen topology of pan-
creatic ductal adenocarcinoma differs from that of normal and chronic pancreatitis. Modern Pathology, 28, 1470–1480.
Erkan, M. (2013a). The role of pancreatic stellate cells in pancreatic cancer. Pancreatology, 13, 106–109.
Erkan, M. (2013b). Antifibrotic therapy in pancreatic diseases. Gut, 62, 1244–1245.
European Association for Study of Liver, Asociacion Latinoamericana para el Estudio del Higado. (2015). EASL-ALEH clinical practice
guidelines: Non-invasive tests for evaluation of liver disease severity and prognosis. Journal of Hepatology, 63, 237–264.
European Medicines Agency. (2017). EMA's final opinion confirms restrictions on use of linear gadolinium agents in body scans.
Fang, M., Yuan, J., Peng, C., & Li, Y. (2014). Collagen as a double-edged sword in tumor progression. Tumour Biology, 35, 2871–2882.
Farrar, C. T., DePeralta, D. K., Day, H., Rietz, T. A., Wei, L., Lauwers, G. Y., … Caravan, P. (2015). 3D molecular MR imaging of liver fibrosis
and response to rapamycin therapy in a bile duct ligation rat model. Journal of Hepatology, 63, 689–696.
Farrar, C. T., Gale, E. M., Kennan, R., Ramsay, I., Masia, R., Arora, G., … Caravan, P. (2018). CM-101: Type I collagen-targeted MR imaging
probe for detection of liver fibrosis. Radiology, 287, 581–589.
Feehally, J., Floege, J., & Johnson, R. (2014). Comprehensive clinical nephrology (5th ed.). Philadelphia, PA: Elsevier.
Fotenos A. (2018). Update on FDA approach to safety issue of gadolinium retention after administration of gadolinium-based contrast agents.
Friedman, S. L. (2015). Clarity and challenges in tissue fibrosis. In K. Nakao, N. Minato, & S. Uemoto (Eds.), Innovative medicine: Basic
research and development (pp. 187–194). Tokyo: Springer.
Friedman, S. L., Sheppard, D., Duffield, J. S., & Violette, S. (2013). Therapy for fibrotic diseases: Nearing the starting line. Science Transla-
tional Medicine, 5 167sr161.
Fryer, E., Wang, L. M., Verrill, C., & Fleming, K. (2013). How often do our liver core biopsies reach current definitions of adequacy? Journal
of Clinical Pathology, 66, 1087–1089.
Fuchs, B. C., Wang, H., Yang, Y., Wei, L., Polasek, M., Schuhle, D. T., … Caravan, P. (2013). Molecular MRI of collagen to diagnose and stage
liver fibrosis. Journal of Hepatology, 59, 992–998.
Gerlinger, M., Rowan, A. J., Horswell, S., Math, M., Larkin, J., Endesfelder, D., … Swanton, C. (2012). Intratumor heterogeneity and
branched evolution revealed by multiregion sequencing. The New England Journal of Medicine, 366, 883–892.
Ghatak, S., Maytin, E. V., Mack, J. A., Hascall, V. C., Atanelishvili, I., Moreno Rodriguez, R., … Misra, S. (2015). Roles of proteoglycans and
glycosaminoglycans in wound healing and fibrosis. International Journal of Cell Biology, 2015, 834893.
Glaus, C., Rossin, R., Welch, M. J., & Bao, G. (2010). In vivo evaluation of (64)Cu-labeled magnetic nanoparticles as a dual-modality PET/MR
imaging agent. Bioconjugate Chemistry, 21, 715–722.
Goodman, J. D., Rozypal, T. L., & Kelly, T. (2003). Seprase, a membrane-bound protease, alleviates the serum growth requirement of human
breast cancer cells. Clinical & Experimental Metastasis, 20, 459–470.
Grossniklaus, H. E. (2013). Progression of ocular melanoma metastasis to the liver: The 2012 Zimmerman lecture. JAMA Ophthalmology,
131, 462–469.
Grossniklaus, H. E., Zhang, Q., You, S., McCarthy, C., Heegaard, S., & Coupland, S. E. (2016). Metastatic ocular melanoma to the liver
exhibits infiltrative and nodular growth patterns. Human Pathology, 57, 165–175.
Gu, L., Li, X., Jiang, J., Guo, G., Wu, H., Wu, M., & Zhu, H. (2018). Stem cell tracking using effective self-assembled peptide-modified super-
paramagnetic nanoparticles. Nanoscale, 10, 15967–15979.
Han, Z., Zhou, Z., Shi, X., Wang, J., Wu, X., Sun, D., … Lu, Z. R. (2015). EDB fibronectin specific peptide for prostate cancer targeting.
Bioconjugate Chemistry, 26, 830–838.
Haqq, J., Howells, L. M., Garcea, G., Metcalfe, M. S., Steward, W. P., & Dennison, A. R. (2014). Pancreatic stellate cells and pancreas cancer:
Current perspectives and future strategies. European Journal of Cancer, 50, 2570–2582.
Heeneman, S., Cleutjens, J. P., Faber, B. C., Creemers, E. E., van Suylen, R. J., Lutgens, E., … Daemen, M. J. (2003). The dynamic extracellu-
lar matrix: Intervention strategies during heart failure and atherosclerosis. The Journal of Pathology, 200, 516–525.
Helm, P. A., Caravan, P., French, B. A., Jacques, V., Shen, L., Xu, Y., … Epstein, F. H. (2008). Postinfarction myocardial scarring in mice:
Molecular MR imaging with use of a collagen-targeting contrast agent. Radiology, 247, 788–796.
Hidalgo, M. (2010). Pancreatic cancer. The New England Journal of Medicine, 362, 1605–1617.
 19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SALARIAN ET AL. 19 of 22

Ibrahim, M. A., Hazhirkarzar, B., Gupta, M., & Dublin, A. B. (2020). Magnetic resonance imaging (MRI) gadolinium. Treasure Island, FL:
StatPearls Publishing.
Jablonska-Trypuc, A., Matejczyk, M., & Rosochacki, S. (2016). Matrix metalloproteinases (MMPs), the main extracellular matrix (ECM)
enzymes in collagen degradation, as a target for anticancer drugs. Journal of Enzyme Inhibition and Medicinal Chemistry, 31, 177–183.
Jeay, S., Pianetti, S., Kagan, H. M., & Sonenshein, G. E. (2003). Lysyl oxidase inhibits ras-mediated transformation by preventing activation
of NF-kappa B. Molecular and Cellular Biology, 23, 2251–2263.
Johnson, G. A., Benveniste, H., Black, R. D., Hedlund, L. W., Maronpot, R. R., & Smith, B. R. (1993). Histology by magnetic resonance
microscopy. Magnetic Resonance Quarterly, 9, 1–30.
Katsuda, S., & Kaji, T. (2003). Atherosclerosis and extracellular matrix. Journal of Atherosclerosis and Thrombosis, 10, 267–274.
Klink, A., Heynens, J., Herranz, B., Lobatto, M. E., Arias, T., Sanders, H. M., … Fayad, Z. A. (2011). In vivo characterization of a new abdomi-
nal aortic aneurysm mouse model with conventional and molecular magnetic resonance imaging. Journal of the American College of Car-
diology, 58, 2522–2530.
Koyama, Y., & Brenner, D. A. (2017). Liver inflammation and fibrosis. The Journal of Clinical Investigation, 127, 55–64.
Kranenburg, A. R., Willems-Widyastuti, A., Moori, W. J., Sterk, P. J., Alagappan, V. K., de Boer, W. I., & Sharma, H. S. (2006). Enhanced
bronchial expression of extracellular matrix proteins in chronic obstructive pulmonary disease. American Journal of Clinical Pathology,
126, 725–735.
Krishnan, S., Otaki, Y., Doris, M., Slipczuk, L., Arnson, Y., Rubeaux, M., … Tamarappoo, B. (2017). Molecular imaging of vulnerable coronary
plaque: A pathophysiologic perspective. Journal of Nuclear Medicine, 58, 359–364.
Leite, A. R., Correa-Giannella, M. L., Dagli, M. L., Fortes, M. A., Vegas, V. M., & Giannella-Neto, D. (2007). Fibronectin and laminin induce
expression of islet cell markers in hepatic oval cells in culture. Cell and Tissue Research, 327, 529–537.
Li, H., Yang, W., Chen, P. W., Alizadeh, H., & Niederkorn, J. Y. (2009). Inhibition of chemokine receptor expression on uveal melanomas by
CXCR4 siRNA and its effect on uveal melanoma liver metastases. Investigative Ophthalmology & Visual Science, 50, 5522–5528.
Li, S., Jiang, J., Zou, J., Qiao, J., Xue, S., Wei, L., … Yang, J. J. (2012). PEGylation of protein-based MRI contrast agents improves relaxivities
and biocompatibilities. Journal of Inorganic Biochemistry, 107, 111–118.
Li, W., Nugent, M. A., Zhao, Y., Chau, A. N., Li, S. J., Chou, I. N., … Kagan, H. M. (2003). Lysyl oxidase oxidizes basic fibroblast growth factor
and inactivates its mitogenic potential. Journal of Cellular Biochemistry, 88, 152–164.
Li, X., Shepard, H. M., Cowell, J. A., Zhao, C., Osgood, R. J., Rosengren, S., … Thompson, C. B. (2018). Parallel accumulation of tumor
hyaluronan, collagen, and other drivers of tumor progression. Clinical Cancer Research, 24, 4798–4807.
Li, Y., Han, Z., Roelle, S., DeSanto, A., Sabatelle, R., Schur, R., & Lu, Z. R. (2017). Synthesis and assessment of peptide Gd-DOTA conjugates
targeting extradomain B fibronectin for magnetic resonance molecular imaging of prostate cancer. Molecular Pharmaceutics, 14,
3906–3915.
Liao, A., Mittal, P., Lawson, D. H., Yang, J. J., Szalai, E., & Grossniklaus, H. E. (2018). Radiologic and histopathologic correlation of different
growth patterns of metastatic uveal melanoma to the liver. Ophthalmology, 125, 597–605.
Lin, J. B., Phillips, E. H., Riggins, T. E., Sangha, G. S., Chakraborty, S., Lee, J. Y., … Goergen, C. J. (2015). Imaging of small animal peripheral
artery disease models: Recent advancements and translational potential. International Journal of Molecular Sciences, 16, 11131–11177.
Liu, X. Y., Liu, R. X., Hou, F., Cui, L. J., Li, C. Y., Chi, C., … Yin, C. H. (2016). Fibronectin expression is critical for liver fibrogenesis in vivo
and in vitro. Molecular Medicine Reports, 14, 3669–3675.
Lunardi, S., Muschel, R. J., & Brunner, T. B. (2014). The stromal compartments in pancreatic cancer: Are there any therapeutic targets? Can-
cer Letters, 343, 147–155.
Mahadevan, D., & Von Hoff, D. D. (2007). Tumor-stroma interactions in pancreatic ductal adenocarcinoma. Molecular Cancer Therapeutics,
6, 1186–1197.
Major, J. L., & Meade, T. J. (2009). Bioresponsive, cell-penetrating, and multimeric MR contrast agents. Accounts of Chemical Research, 42,
893–903.
Mak, K. M., Chen, L. L., & Lee, T. F. (2013). Codistribution of collagen type IV and laminin in liver fibrosis of elderly cadavers: Immunohis-
tochemical marker of perisinusoidal basement membrane formation. The Anatomical Record (Hoboken), 296, 953–964.
Mak, K. M., & Mei, R. (2017). Basement membrane type IV collagen and laminin: An overview of their biology and value as fibrosis bio-
markers of liver disease. The Anatomical Record (Hoboken), 300, 1371–1390.
Makowski, M. R., Wiethoff, A. J., Blume, U., Cuello, F., Warley, A., Jansen, C. H., … Botnar, R. M. (2011). Assessment of atherosclerotic
plaque burden with an elastin-specific magnetic resonance contrast agent. Nature Medicine, 17, 383–388.
Malik, G., Knowles, L. M., Dhir, R., Xu, S., Yang, S., Ruoslahti, E., & Pilch, J. (2010). Plasma fibronectin promotes lung metastasis by contri-
butions to fibrin clots and tumor cell invasion. Cancer Research, 70, 4327–4334.
Mankoff, D. A. (2007). A definition of molecular imaging. Journal of Nuclear Medicine, 48 18N, 21N.
Modol, T., Brice, N., Ruiz de Galarreta, M., Garcia Garzon, A., Iraburu, M. J., Martinez-Irujo, J. J., & Lopez-Zabalza, M. J. (2015). Fibronectin
peptides as potential regulators of hepatic fibrosis through apoptosis of hepatic stellate cells. Journal of Cellular Physiology, 230, 546–553.
Mouw, J. K., Ou, G., & Weaver, V. M. (2014). Extracellular matrix assembly: A multiscale deconstruction. Nature Reviews. Molecular Cell
Biology, 15, 771–785.
Murray, L. A. (2015). Editorial: The cell types of fibrosis. Frontiers in Pharmacology, 6, 311.
Myers, R. P., Fong, A., & Shaheen, A. A. (2008). Utilization rates, complications and costs of percutaneous liver biopsy: A population-based
study including 4275 biopsies. Liver International, 28, 705–712.
 19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
20 of 22 SALARIAN ET AL.

Myllyharju, J., & Kivirikko, K. I. (2004). Collagens, modifying enzymes and their mutations in humans, flies and worms. Trends in Genetics,
20, 33–43.
Nair, S. A., Kolodziej, A. F., Bhole, G., Greenfield, M. T., McMurry, T. J., & Caravan, P. (2008). Monovalent and bivalent fibrin-specific MRI
contrast agents for detection of thrombus. Angewandte Chemie (International Ed. in English), 47, 4918–4921.
Okamura, H., Pisani, L. J., Dalal, A. R., Emrich, F., Dake, B. A., Arakawa, M., … Fischbein, M. P. (2014). Assessment of elastin deficit in a
Marfan mouse aneurysm model using an elastin-specific magnetic resonance imaging contrast agent. Circulation. Cardiovascular Imag-
ing, 7, 690–696.
Olive, K. P., Jacobetz, M. A., Davidson, C. J., Gopinathan, A., McIntyre, D., Honess, D., … Tuveson, D. A. (2009). Inhibition of hedgehog sig-
naling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science, 324, 1457–1461.
Oliveira, B. L., & Caravan, P. (2017). Peptide-based fibrin-targeting probes for thrombus imaging. Dalton Transactions, 46, 14488–14508.
Overoye-Chan, K., Koerner, S., Looby, R. J., Kolodziej, A. F., Zech, S. G., Deng, Q., … Caravan, P. (2008). EP-2104R: A fibrin-specific
gadolinium-based MRI contrast agent for detection of thrombus. Journal of the American Chemical Society, 130, 6025–6039.
Pankova, D., Chen, Y., Terajima, M., Schliekelman, M. J., Baird, B. N., Fahrenholtz, M., … Kurie, J. M. (2016). Cancer-associated fibroblasts
induce a collagen cross-link switch in tumor stroma. Molecular Cancer Research, 14, 287–295.
Paszek, M. J., Zahir, N., Johnson, K. R., Lakins, J. N., Rozenberg, G. I., Gefen, A., … Weaver, V. M. (2005). Tensional homeostasis and the
malignant phenotype. Cancer Cell, 8, 241–254.
Perez-Rico, C., Pascual, G., Sotomayor, S., Asunsolo, A., Cifuentes, A., Garcia-Honduvilla, N., & Bujan, J. (2014). Elastin development-
associated extracellular matrix constituents of subepithelial connective tissue in human pterygium. Investigative Ophthalmology & Visual
Science, 55, 6309–6318.
Perez-Rico, C., Pascual, G., Sotomayor, S., Montes-Mollon, M. A., Trejo, C., Sasaki, T., … Bujan, J. (2011). Tropoelastin and fibulin over-
expression in the subepithelial connective tissue of human pterygium. American Journal of Ophthalmology, 151, 44–52.
Phinikaridou, A., Lacerda, S., Lavin, B., Andia, M. E., Smith, A., Saha, P., & Botnar, R. M. (2018). Tropoelastin: A novel marker for plaque
progression and instability. Circulation. Cardiovascular Imaging, 11.
Polasek, M., Fuchs, B. C., Uppal, R., Schuhle, D. T., Alford, J. K., Loving, G. S., … Caravan, P. (2012). Molecular MR imaging of liver fibrosis:
A feasibility study using rat and mouse models. Journal of Hepatology, 57, 549–555.
Popov, Y., & Schuppan, D. (2009). Targeting liver fibrosis: Strategies for development and validation of antifibrotic therapies. Hepatology, 50,
1294–1306.
Protti, A., Lavin, B., Dong, X., Lorrio, S., Robinson, S., Onthank, D., … Botnar, R. M. (2015). Assessment of myocardial remodeling using an
elastin/tropoelastin specific agent with high field magnetic resonance imaging (MRI). Journal of the American Heart Association, 4,
e001851.
Provenzano, P. P., Cuevas, C., Chang, A. E., Goel, V. K., Von Hoff, D. D., & Hingorani, S. R. (2012). Enzymatic targeting of the stroma ablates
physical barriers to treatment of pancreatic ductal adenocarcinoma. Cancer Cell, 21, 418–429.
Provenzano, P. P., & Hingorani, S. R. (2013). Hyaluronan, fluid pressure, and stromal resistance in pancreas cancer. British Journal of Cancer,
108, 1–8.
Provenzano, P. P., Inman, D. R., Eliceiri, K. W., Knittel, J. G., Yan, L., Rueden, C. T., … Keely, P. J. (2008). Collagen density promotes mam-
mary tumor initiation and progression. BMC Medicine, 6, 11.
Pu, F., Qiao, J., Xue, S., Yang, H., Patel, A., Wei, L., … Yang, J. J. (2015). GRPR-targeted protein contrast agents for molecular imaging of
receptor expression in cancers by MRI. Scientific Reports, 5, 16214.
Pu, F., Salarian, M., Xue, S., Qiao, J., Feng, J., Tan, S., … Yang, J. J. (2016). Prostate-specific membrane antigen targeted protein contrast
agents for molecular imaging of prostate cancer by MRI. Nanoscale, 8, 12668–12682.
Pu, F., Xue, S., Qiao, J., Patel, A., & Yang, J. J. (2016). Towards the molecular imaging of prostate cancer biomarkers using protein-based
MRI contrast agents. Current Protein & Peptide Science, 17, 519–533.
Qiao, J., Li, S., Wei, L., Jiang, J., Long, R., Mao, H., … Yang, J. J. (2011). HER2 targeted molecular MR imaging using a de novo designed pro-
tein contrast agent. PLoS ONE, 6, e18103.
Qiao, J., Xue, S., Pu, F., White, N., Jiang, J., Liu, Z. R., & Yang, J. J. (2014). Molecular imaging of EGFR/HER2 cancer biomarkers by protein
MRI contrast agents. Journal of Biological Inorganic Chemistry, 19, 259–270.
Ricard-Blum, S. (2011). The collagen family. Cold Spring Harbor Perspectives in Biology, 3, a004978.
Ricard-Blum, S., & Ruggiero, F. (2005). The collagen superfamily: From the extracellular matrix to the cell membrane. Pathologie Biologie
(Paris), 53, 430–442.
Robertson, E. G., & Baxter, G. (2011). Tumour seeding following percutaneous needle biopsy: The real story! Clinical Radiology, 66,
1007–1014.
Rotman, Y., & Sanyal, A. J. (2017). Current and upcoming pharmacotherapy for non-alcoholic fatty liver disease. Gut, 66, 180–190.
Rousselet, M. C., Michalak, S., Dupre, F., Croue, A., Bedossa, P., Saint-Andre, J. P., … Hepatitis, N. (2005). Sources of variability in histologi-
cal scoring of chronic viral hepatitis. Hepatology, 41, 257–264.
Salarian, M., Turaga, R. C., Xue, S., Nezafati, M., Hekmatyar, K., Qiao, J., … Yang, J. J. (2019). Early detection and staging of chronic liver dis-
eases with a protein MRI contrast agent. Nature Communications, 10, 4777.
Salarian, M., Yang, H., Turaga, R. C., Tan, S., Qiao, J., Xue, S., … Yang, J. J. (2019). Precision detection of liver metastasis by collagen-targeted
protein MRI contrast agent. Biomaterials, 224, 119478.
 19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SALARIAN ET AL. 21 of 22

Sanders, H. M., Strijkers, G. J., Mulder, W. J., Huinink, H. P., Erich, S. J., Adan, O. C., … Nicolay, K. (2009). Morphology, binding behavior
and MR-properties of paramagnetic collagen-binding liposomes. Contrast Media & Molecular Imaging, 4, 81–88.
Sanyal, S., Marckmann, P., Scherer, S., & Abraham, J. L. (2011). Multiorgan gadolinium (Gd) deposition and fibrosis in a patient with
nephrogenic systemic fibrosis – An autopsy-based review. Nephrology, Dialysis, Transplantation, 26, 3616–3626.
Schwarzbauer, J. E. (1991). Identification of the fibronectin sequences required for assembly of a fibrillar matrix. The Journal of Cell Biology,
113, 1463–1473.
Schwarzbauer, J. E., & DeSimone, D. W. (2011). Fibronectins, their fibrillogenesis, and in vivo functions. Cold Spring Harbor Perspectives in
Biology, 3.
Scodellaro, R., Bouzin, M., Mingozzi, F., D'Alfonso, L., Granucci, F., Collini, M., … Sironi, L. (2019). Whole-section tumor micro-architecture
analysis by a two-dimensional phasor-based approach applied to polarization-dependent second harmonic imaging. Frontiers in Oncol-
ogy, 9, 527.
Shields, M. A., Dangi-Garimella, S., Redig, A. J., & Munshi, H. G. (2012). Biochemical role of the collagen-rich tumour microenvironment in
pancreatic cancer progression. The Biochemical Journal, 441, 541–552.
Shoulders, M. D., & Raines, R. T. (2009). Collagen structure and stability. Annual Review of Biochemistry, 78, 929–958.
Singh, P., Carraher, C., & Schwarzbauer, J. E. (2010). Assembly of fibronectin extracellular matrix. Annual Review of Cell and Developmental
Biology, 26, 397–419.
Sonbol, H. S. (2018). Extracellular matrix remodeling in human disease. Journal of Microscopy and Ultrastructure, 6, 123–128.
Spuentrup, E., Botnar, R. M., Wiethoff, A. J., Ibrahim, T., Kelle, S., Katoh, M., … Maintz, D. (2008). MR imaging of thrombi using EP-2104R,
a fibrin-specific contrast agent: Initial results in patients. European Radiology, 18, 1995–2005.
Spuentrup, E., Buecker, A., Katoh, M., Wiethoff, A. J., Parsons, E. C., Jr., Botnar, R. M., … Gunther, R. W. (2005). Molecular magnetic reso-
nance imaging of coronary thrombosis and pulmonary emboli with a novel fibrin-targeted contrast agent. Circulation, 111, 1377–1382.
Spuentrup, E., Fausten, B., Kinzel, S., Wiethoff, A. J., Botnar, R. M., Graham, P. B., … Buecker, A. (2005). Molecular magnetic resonance
imaging of atrial clots in a swine model. Circulation, 112, 396–399.
Spuentrup, E., Katoh, M., Wiethoff, A. J., Parsons, E. C., Jr., Botnar, R. M., Mahnken, A. H., … Buecker, A. (2005). Molecular magnetic reso-
nance imaging of pulmonary emboli with a fibrin-specific contrast agent. American Journal of Respiratory and Critical Care Medicine,
172, 494–500.
Spuentrup, E., Ruhl, K. M., Botnar, R. M., Wiethoff, A. J., Buhl, A., Jacques, V., … Caravan, P. (2009). Molecular magnetic resonance imaging
of myocardial perfusion with EP-3600, a collagen-specific contrast agent: Initial feasibility study in a swine model. Circulation, 119,
1768–1775.
Stracke, C. P., Katoh, M., Wiethoff, A. J., Parsons, E. C., Spangenberg, P., & Spuntrup, E. (2007). Molecular MRI of cerebral venous sinus
thrombosis using a new fibrin-specific MR contrast agent. Stroke, 38, 1476–1481.
Sun, Q., Baues, M., Klinkhammer, B. M., Ehling, J., Djudjaj, S., Drude, N. I., … Boor, P. (2019). Elastin imaging enables noninvasive staging
and treatment monitoring of kidney fibrosis. Science Translational Medicine, 11, eaat4865.
Sun, Y., Ding, F., Chen, Z., Zhang, R., Li, C., Xu, Y., … Stang, P. J. (2019). Melanin-dot-mediated delivery of metallacycle for NIR-
II/photoacoustic dual-modal imaging-guided chemo-photothermal synergistic therapy. Proceedings of the National Academy of Sciences
of the United States of America, 116, 16729–16735.
Sun, Y., Ding, F., Zhou, Z., Li, C., Pu, M., Xu, Y., … Stang, P. J. (2019). Rhomboidal Pt(II) metallacycle-based NIR-II theranostic nanoprobe
for tumor diagnosis and image-guided therapy. Proceedings of the National Academy of Sciences of the United States of America, 116,
1968–1973.
Thakral, C., & Abraham, J. L. (2009). Gadolinium-induced nephrogenic systemic fibrosis is associated with insoluble Gd deposits in tissues:
in vivo transmetallation confirmed by microanalysis. Journal of Cutaneous Pathology, 36, 1244–1254.
Toczek, J., & Sadeghi, M. M. (2016). Molecular imaging concepts. Journal of Nuclear Cardiology, 23, 271–273.
Tripepi, M., Capuana, F., Gianolio, E., Kock, F. V. C., Pagoto, A., Stefania, R., … Aime, S. (2018). Synthesis of high relaxivity gadolinium
AAZTA tetramers as building blocks for bioconjugation. Bioconjugate Chemistry, 29, 1428–1437.
Uppal, R., Ay, I., Dai, G., Kim, Y. R., Sorensen, A. G., & Caravan, P. (2010). Molecular MRI of intracranial thrombus in a rat ischemic stroke
model. Stroke, 41, 1271–1277.
Uppal, R., Catana, C., Ay, I., Benner, T., Sorensen, A. G., & Caravan, P. (2011). Bimodal thrombus imaging: Simultaneous PET/MR imaging
with a fibrin-targeted dual PET/MR probe – Feasibility study in rat model. Radiology, 258, 812–820.
Vithanarachchi, S. M., & Allen, M. J. (2012). Strategies for target-specific contrast agents for magnetic resonance imaging. Curr Mol Imaging,
1, 12–25.
Vymazal, J., Spuentrup, E., Cardenas-Molina, G., Wiethoff, A. J., Hartmann, M. G., Caravan, P., & Parsons, E. C., Jr. (2009). Thrombus imag-
ing with fibrin-specific gadolinium-based MR contrast agent EP-2104R: Results of a phase II clinical study of feasibility. Investigative
Radiology, 44, 697–704.
Wadas, T. J., Wong, E. H., Weisman, G. R., & Anderson, C. J. (2010). Coordinating radiometals of copper, gallium, indium, yttrium, and zir-
conium for PET and SPECT imaging of disease. Chemical Reviews, 110, 2858–2902.
Walker, C., Mojares, E., & Del Rio Hernandez, A. (2018). Role of extracellular matrix in development and cancer progression. International
Journal of Molecular Sciences, 19.
Wang, B., Sun, Y., Zhou, J., Wu, X., Chen, S., Shi, Y., … You, H. (2019). SHG/TPEF-based image technology improves liver fibrosis assess-
ment of minimally sized needle biopsies. Hepatology International, 13, 501–509.
 19390041, 2020, 4, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wnan.1622 by Johns Hopkins University, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
22 of 22 SALARIAN ET AL.

Wei, L., Li, S., Yang, J., Ye, Y., Zou, J., Wang, L., … Liu, Z. R. (2011). Protein-based MRI contrast agents for molecular imaging of prostate
cancer. Molecular Imaging and Biology, 13, 416–423.
Wynn, T. A. (2008). Cellular and molecular mechanisms of fibrosis. The Journal of Pathology, 214, 199–210.
Xue, S., Qiao, J., Jiang, J., Hubbard, K., White, N., Wei, L., … Yang, J. J. (2014). Design of ProCAs (protein-based Gd(3+) MRI contrast
agents) with high dose efficiency and capability for molecular imaging of cancer biomarkers. Medicinal Research Reviews, 34, 1070–1099.
Xue, S., Qiao, J., Pu, F., Cameron, M., & Yang, J. J. (2013). Design of a novel class of protein-based magnetic resonance imaging contrast
agents for the molecular imaging of cancer biomarkers. WIREs Nanomedicine and Nanobiotechnology, 5, 163–179.
Xue, S., Yang, H., Qiao, J., Pu, F., Jiang, J., Hubbard, K., … Yang, J. J. (2015). Protein MRI contrast agent with unprecedented metal selectiv-
ity and sensitivity for liver cancer imaging. Proceedings of the National Academy of Sciences of the United States of America, 112,
6607–6612.
Yang, H., & Grossniklaus, H. E. (2006). Combined immunologic and anti-angiogenic therapy reduces hepatic micrometastases in a murine
ocular melanoma model. Current Eye Research, 31, 557–562.
Yang, J. J., Yang, J., Wei, L., Zurkiya, O., Yang, W., Li, S., … Liu, Z. R. (2008). Rational design of protein-based MRI contrast agents. Journal
of the American Chemical Society, 130, 9260–9267.
Yao, E. S., Zhang, H., Chen, Y. Y., Lee, B., Chew, K., Moore, D., & Park, C. (2007). Increased beta1 integrin is associated with decreased sur-
vival in invasive breast cancer. Cancer Research, 67, 659–664.
Ye, F., Wu, X., Jeong, E. K., Jia, Z., Yang, T., Parker, D., & Lu, Z. R. (2008). A peptide targeted contrast agent specific to fibrin-fibronectin
complexes for cancer molecular imaging with MRI. Bioconjugate Chemistry, 19, 2300–2303.
Zhang, P., Cui, Y., Anderson, C. F., Zhang, C., Li, Y., Wang, R., & Cui, H. (2018). Peptide-based nanoprobes for molecular imaging and dis-
ease diagnostics. Chemical Society Reviews, 47, 3490–3529.
Zhang, R., Xu, Y., Zhang, Y., Kim, H. S., Sharma, A., Gao, J., … Sun, Y. (2019). Rational design of a multifunctional molecular dye for dual-
modal NIR-II/photoacoustic imaging and photothermal therapy. Chemical Science, 10, 8348–8353.
Zhang, Z., Kolodziej, A. F., Qi, J., Nair, S. A., Wang, X., Case, A. W., … Caravan, P. (2010). Effect of peptide-chelate architecture on metabolic
stability of peptide-based MRI contrast agents. New Journal of Chemistry, 2010, 611–616.
Zhou, Z., Qutaish, M., Han, Z., Schur, R. M., Liu, Y., Wilson, D. L., & Lu, Z. R. (2015). MRI detection of breast cancer micrometastases with
a fibronectin-targeting contrast agent. Nature Communications, 6, 7984.
Zhou, Z., Wu, X., Kresak, A., Griswold, M., & Lu, Z. R. (2013). Peptide targeted tripod macrocyclic Gd(III) chelates for cancer molecular
MRI. Biomaterials, 34, 7683–7693.
Zong, Y., Xu, Y., Liang, X., Keene, D. R., Hook, A., Gurusiddappa, S., … Narayana, S. V. (2005). A ‘Collagen Hug’ model for Staphylococcus
aureus CNA binding to collagen. The EMBO Journal, 24, 4224–4236.

How to cite this article: Salarian M, Ibhagui OY, Yang JJ. Molecular imaging of extracellular matrix proteins
with targeted probes using magnetic resonance imaging. WIREs Nanomed Nanobiotechnol. 2020;12:e1622.
https://doi.org/10.1002/wnan.1622