Gadolinium‐containing carbon nanomaterials for magnetic resonance imaging: Trends and challenges

Abstract Gadolinium‐containing carbon nanomaterials are a new class of contrast agent for magnetic resonance imaging. They are characterized by a superior proton relaxivity to any current commercial gadolinium contrast agent and offer the possibility to design multifunctional contrasts. Intense efforts have been made to develop these nanomaterials because of their potential for better results than the available gadolinium contrast agents. The aim of the present work is to provide a review of the advances in research on gadolinium‐containing carbon nanomaterials and their advantages over conventional gadolinium contrast agents. Due to their enhanced proton relaxivity, they can provide a reliable imaging contrast for cells, tissues or organs with much smaller doses than currently used in clinical practice, thus leading to reduced toxicity (as shown by cytotoxicity and biodistribution studies). Their active targeting capability allows for improved MRI of molecular or cellular targets, overcoming the limited labelling capability of available contrast agents (restricted to physiological irregularities during pathological conditions). Their potential of multifunctionality encompasses multimodal imaging and the combination of imaging and therapy.


| INTRODUC TI ON
Magnetic resonance imaging (MRI) is one of the most important tools for clinical imaging. Apart from its unlimited tissue penetration, MRI offers the advantages of being non-invasive and producing zero ionizing radiation. Because of being able to distinguish tumours from healthy tissue, it is considered essential for the diagnosis and prognosis of cancer as well as for surveying the efficacy of the pharmacotherapy. 1 Additionally, it is used for the preparation and guidance of surgical procedures (eg brain tumour resections) 2 and for basic biomedical research.
An MRI of the human body is based on signals produced by water protons under a strong magnetic field. 3 Since most of the human body is made up of water molecules, MRI can identify a given organ or tissue structure by detecting water proton density, proton velocity, and the longitudinal (T1) and transverse (T2) relaxation times. 4 When the intrinsic contrast between diseased and healthy tissues is too low for an accurate diagnosis, 5 contrast agents are intravenously administered. These chemical entities improve the visualization of tissues or organs by increasing the relative difference between the signal intensity. 6 They improve MRI contrast by shortening the longitudinal (T1) and/or transverse (T2) relaxation times of water protons.
MRI contrast agents are classified as T1 or T2 according to the type of relaxation time that is improved. T1 predominantly reduces the longitudinal relaxation time of water protons, thus enhancing a positive (bright) contrast in imaging. Contrarily, T2 primarily diminishes the transverse relaxation time, improving the negative (dark) contrast. 7,8 Regarding the T1-type, the main medical imaging agents for the visualization of tissues and organs contain gadolinium (III) [Gd (III)]. This metal ion couples a large magnetic moment with a long electron spin relaxation time. 9 Although Gd(III) complexes have been administered to more than 100 million patients and demonstrated an extraordinarily positive safety record, 10 it is necessary to develop new contrast agents that improve diagnosis accuracy. 11 Gadolinium-containing carbon nanomaterials, a new class of T1 contrast agents, rank among the most potent in terms of proton relaxivity (r 1 ). They induce 10-to 90-fold greater relaxivity 12,13 than the available gadolinium-based agents. For example, the relaxivity induced by Gd(DTPA) or Gd(DOTA) is at about 4 mM −1 s −1 while for gadofullerenes is around r 1 = 47.0 ± 1.0 mM −1 s −1 . 11 The new gadolinium contrast consists of carbon nanomaterials with gadolinium ions confined in their inner cavities or attached by ligands to functional groups on their external surface, tips or edges. The Figure 1 shows the principal four types of contrast agents: gadofullerenes, gadonanotubes, gadonanodiamonds and gadographene.
Compared to commercial products, the new contrast agents show several advantages. Recent reviews have discussed the advances of gadolinium-containing carbon nanomaterials in relation to the methods of synthesis as well as their chemical and physical properties. Each review has focused on a certain type of nanomaterial, such as gadonanotubes [14][15][16] or gadofullerenes. [17][18][19][20][21][22] The present review consists of an overview of gadolinium-containing carbon nanomaterials, comparing them with conventional gadolinium contrast agents and considering their advantages, disadvantages, cytotoxicity and biodistribution.

| P OTENTIAL ADVANTAG E S
There are several important advantages afforded by gadoliniumcontaining carbon nanomaterials compared to commercial contrast agents ( Figure 2). Firstly, their greater proton relaxivity allows for the attainment of reliable images with a lower dose. Hence, less Gd(III) ions are needed (<10-fold less than the current clinical dose) to produce the same brightness as available contrast agents. 23  compared to the current contrast agents. They reported the first T1-weighted MRI phantoms with gadonanodiamonds ( Figure 3C). In 2012, Shen et al 38 showed the high relaxivity produced by gadographene, demonstrated by the first T1-weighted MRI phantoms with this contrast agent ( Figure 3D).
Although the working mechanism for the superior relaxivity of gadolinium-containing carbon nanomaterials is still not well understood, it is often described by the Solomon, Bloembergen and Morgan (SBM) theory. This theory, initially developed to explain the proton relaxivity of gadolinium chelated contrasts, provides insights into gadolinium-containing carbon nanomaterials. Several parameters of the SBM theory can help to account for the observed high relaxivity of the new carbon contrast. These include the number of water molecules co-ordinated to the paramagnetic centre (q), the rotational correlation time (TR) and the mean residence life-time of the co-ordinate waters (TM).
The number of water molecules co-ordinated is considered one of the most important. Gd(III) is a paramagnetic element with a fluctuating magnetic field that interacts with nearby water molecules and thus shortens their T1 and T2 relaxation times and improves the bright contrast in MR imaging. 39 The Gd(III) complex is hydrated by three different types of water molecules that may be affected by the magnetic field ( Figure 4). According to the SBM theory, proton relaxivity enhancement is more pronounced for water molecules directly co-ordinated to the paramagnetic centre (in the inner sphere) than those at a greater distance (in the second and outer sphere). These water molecules play a key role in transmitting the paramagnetic effect towards the bulk solvent and therefore in improving the contrast. Commercial Gd(III) contrast agents usually have one co-ordinate water molecule (q = 1) and a relaxivity of approximately of four. For example, Gd(DTPA) displays an r 1 value of 4.30 mM −1 s −1 . Gadonanotubes, on the other hand, reportedly contain a large number of co-ordinated water molecules per Gd(III) (q = 9), as suggested by X-ray absorption spectroscopic analysis, 40 which may explain their elevated relaxivity compared to commercial gadolinium chelated contrast agents. The q value has also been employed to account for the superior performance of gadofullerenes vs commercial agents. 12 The paramagnetic properties of Gd(III) are considered to be conserved and transferred to the fullerene cage in gadofullerenes, where the Gd(III) ion is encapsulated. 41,42 Consequently, there is an enhanced surface area accessible to water molecules, allowing a larger number of them to have direct contact with the paramagnetic centre and undergo the transmission of relaxivity. For gadofullerenes and gadonanotubes, it was reported a second sphere-like mechanism also plays a key role. 43 This second-sphere like is originated by a large number of water molecules bounded to the F I G U R E 2 Schematic illustration of the major biological applications of gadolinium-containing carbon nanomaterials. The chemical and physical properties of carbon nanomaterials let the design of new MRI contrast agents for multimodal imaging, cell tracking, tumour imaging and the combination of diagnosis and therapy functional groups present in the carbon nanostructure surface, which increases the probability of waters protons to be exchanged. 44,45 The SBM theory also consider the rotational correlation time (TR), which refers to the tumbling rate of the contrast agents. 39 The optimal TR value for a boosted relaxivity is in the range of nanoseconds. 37 Small gadolinium contrasts agents tumble too fast, near the tenths of picoseconds, and in order to decrease their tumbling rate they are usually coupled to polymers of high molecular weight. 35  On the other hand, it was found the carbon structure also plays a key role for water proton relaxation. Moghaddam et al, 48 found the carbon-based radical centres that are supported by the polynuclear aromatic structure contribute at low magnetic fields (<1 MHz).
Until now, several parameters have described the relaxivity of gadolinium-containing carbon materials, however, there is a necessity to develop new theoretical approaches for a most robust description [for a more comprehensive review, see 15,40,49,50 ].

| Easy targeting of the contrast agent towards specific cells, tissues or molecules
Nowadays, research on non-invasive imaging focuses on the development of tools for detecting biomarkers, which are unique can diffuse freely and accumulate in the tumour, thereby facilitating MR imaging. 52 Contrarily, contrast agents are not able to cross the blood-brain barrier in healthy tissue.
Another disadvantage of MRI is the inability of most contrast agents to cross intact membranes, restricting them to the extracellular space and thus making it impossible for them to target intracellular biomarkers. 53 To overcome these disadvantages, scientist has focus on the development of new contrast agents to improve the MRI potential for the detection of biomarkers at the cellular or molecular level. In particular, the efforts have been focused on 'active targeting' concept, which involve the binding of certain ligands to contrast agents for specific homing 54 The ligands are selected based on their ability to bind to biomarkers in the target cells (eg membrane receptors overexpressed on the surface of cancer cells), allowing for the uptake and retention of the contrast agent and as a consequence the imaging of the molecular targets.
The active targeting concept inspired the development of gadolinium-based carbon nanomaterials, in which single amino acids, small peptides, cyclic peptides, proteins and organic molecules are integrated as ligands to target specific cells. For instance, Shu et al 26 worked with antibodies able to recognize the green fluorescent protein. These antibodies were bound to carboxylic functional groups of gadofullerenes and the authors suggested that this strategy can be extended to other biomolecules or endohedral fullerenes.
Fillmore et al 27 reported the covalent binding of the cytokine interleukin-13 (IL-13) peptide to the carboxylic groups on gadofullerenes. They selected IL-13 because of its high affinity for the IL-13Rα2 receptor, which is overexpressed on human glioma cells.
When comparing gadofullerenes with and without the IL-13 functionalization, the former displayed a 64-fold greater in vitro uptake. The authors also actively targeted a tumour in mouse brain (implanted by the inoculation of U87 cells) with functionalized gadofullerenes and could achieve MR imaging. Years later, they actively targeted a brain tumour with gadofullerenes bearing amino groups (rather than carboxylic groups) for the covalent binding of IL-13. 23 This conjugate system produced images with a sharp definition of the tumour, unlike the low-quality tumour outline obtained with Magnevist (the control) ( Figure 5). Since active targeting improved the capacity of MRI to detect tumours, the concentration employed was up to 50% lower than that of traditional control contrast agents. 23 Gadofullerenes have been modified with folic acid as well, a ligand with high affinity for its cell membrane receptor, which is over- In summary, specific functionalization of gadofullerenes with high-affinity ligands by biomarkers has enabled the active targeting of tumours at a cellular and molecular level, leading to increased MRI capability.

| Potential for the design of multifunctional nanoprobes
A multifunctional probe is a contrast agent that serves multiple im-   The cell internalization of gadonanotubes has also been described.

| IN VITRO AND ANIMAL MODEL SAFE T Y A SS E SS MENTS
The advantages of gadolinium-containing carbon nanomaterials over conventional contrast agents should certainly have an impact on several areas of medicine, especially imaging and therapy for cancer patients. However, it is necessary to assure the safety of nanoparticles (NPs) interacting with the organism at the cellular and systemic level. Hence, the cytotoxicity and biodistribution of gadolinium-containing carbon nanomaterials are presently explored to provide insights into their effects on biological systems.

| Cytotoxicity
One of the main concerns with the medical applications of carbon nanomaterials containing gadolinium ions is their cytotoxicity, be-

| In vivo toxicity and biodistribution
In vivo studies are indispensable for determining the efficacy and toxicity of gadolinium-containing carbon nanomaterials. They may help to determine how these new contrast agents are biodistributed, what is the clearance mechanisms, the toxicity related with the materials, the maximum tolerable doses, and the assessment of important parameters that may help to determine whether the contrasts are safety for clinical applicability. Table 1 summary the major biological findings from in vivo studies of gadofullerenes and gadonanotubes.
Until now, most of the revised studies were focused to prove the potential application of the contrast agents and complementary efforts were done to understand their toxicity. As can be appreciated in Table 1, most of the studies reported no acute effects, and just three studies described gadonanotubes and gadofullerenes may promote inflammation and effects at genomic level. 76,81,82 However, due to the well-known toxicity of carbon nanomaterials and the toxicity of free gadolinium ions, more studies are needed to clarify the clinical application. For instance, previous works reported carbon nanotubes and fullerenes may promote adverse effects, including inflammation, oxidative stress, DNA damage and mutation, malignant transformation, the formation of granulomas and interstitial fibrosis. 79,83,84 Also, owing the toxicity of free gadolinium ions, it is essential to carry out bioavailability studies by their potential dissociation from the carbon nanostructure. It takes significance because even metal impurities can be released from the carbon nanotube structure despite their apparent encapsulation by carbon. 85 Particularly, the concern emerges by the presence of gadolinium in the brain of patients that received repeated administration of chelated gadolinium contrast agents for MRI. 10,86 The dechelation of free Gd(III) ions from unstable contrast agents is considered the main cause for the observed accumulation. 87  In terms of biodistribution, most of the gadolinium-containing carbon nanomaterials tend to be entrapped by the mononuclear phagocyte system, a network of immune and architectural cells that removes foreign material from the bloodstream. 88 The system encompasses monocytes of the blood, macrophages in connective tissue, lymphoid organs, bone marrow, bone, liver and lung. 88 92 The hepatic accumulation of carbon nanotubes is associated with the induction of a local immune response and liver damage. 93 The accumulation in kidney may induce toxicity due increased oxidative stress, mitochondrial damage or DNA damage. 94,95 In spleen, the accumulation of carbon nanotubes has been associated with immunotoxicity. 96 As can be appreciated, carbon nanotubes and fullerenes are associated with toxic effects, however, also a number of reports

| CON CLUS ION
A critical evaluation was conducted of the advantages of gadolinium-containing carbon nanomaterials over conventional chelated gadolinium contrast agents. Their enhanced relaxivity allows for reliable imaging of cellular and molecular biomarkers with a lower dose of Gd(III), thus reducing the risk of toxicity from free Gd(III) ions. The physical and chemical properties of carbon nanomaterials have enabled the fabrication of contrast agents with multifunctionality as well as with active targeting capability, which encompasses multimodal imaging and the combination of imaging with therapy with high specificity.
Nowadays, there are intense efforts to design new T1 MRI nanoprobes based on gadolinium-containing carbon nanomaterials. However, several issues should be overcome, the production of mono-disperse nanomaterials with controlled size and a homogeneous gadolinium load is still in its early stages, and require better and standardized synthesis methods.
The new contrast must be tested with standard characterization techniques and valid methods to establish the proper relationship between the characteristics of the nanomaterial and its probable toxicity in order to understand its behaviour in living systems. Also, more preclinical studies as biodistribution, biocompatibility, pharmacokinetics and long-term stability are needed to clarify the potential clinical use of these nanomaterials.

ACK N OWLED G EM ENTS
This work was supported by the National Autonomous University of Mexico (UNAM; Grant DGAPA-IN104919).

CO N FLI C T O F I NTE R E S T
No conflict of interest exits in the submission of this manuscript.

AUTH O R CO NTR I B UTI O N
ARG performed the literature research and drafted the manuscript; MR, PGL, LAM and VAB revised the manuscript, drafted the manuscript, edited the final version and gave the final approval for the article to be published.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article.