The advent of magnetic resonance imaging (MRI) has provided scientists and clinicians with a powerful tool to acquire in vivo
images of anatomy and physiology in whole animals and humans. It is routinely used in the clinic for diagnostic imaging and has the advantage of providing exquisite anatomic resolution, routinely down to 0.5 to 1 mm. The signal-to-noise ratios in an MR image depend on the density of protons present in the region of interest and the degree of polarization of the nuclear spin states. When placed in a magnetic field, a slight majority of the magnetic moments of protons will orient in the direction of the magnetic field and precess at a resonance frequency, known as the Larmor frequency, related to the strength of the magnetic field. Relaxation is measured in two directions, longitudinal and transverse. Longitudinal or spin-lattice relaxation is defined by the time constant T1 and occurs in the direction of the main magnetic field. Signals related to T1 relaxation are obtained after excitation by an RF pulse at the Larmor frequency as the proton’s dipole moment vector begins to realign or relax back to its ground state of alignment with the main magnetic field. Transverse or spin-spin relaxation corresponds to vector dephasing in the plane perpendicular to the main magnetic field and is characterized by T2. T1 represents the time required for the magnetization vector to be restored to 63% of its original magnitude and T2, a 37% decrease in net signal. T2 is always shorter than T1. Inhomogeneity in the static magnetic field and spin-spin relaxation has an effect on the transverse magnetization and is characterized by:
’ is a time constant arising from magnetic field inhomogeneity and T2
* is the spin-spin time constant that takes into account these issues. T2
* is always less than T2
. Tissue types vary in their relaxation properties, and thus MRI is used to reconstruct images of structures such as organs and lesions and to evaluate perfusion and flow-related abnormalities.
MRI has proven to be extremely successful in the clinical environment. However, all technologies have their limits. In the case of MRI, signal intensity is dependent on several parameters such as proton density, T1, T2 and T2* relaxation rates, magnetic field strength, and pulse sequence. While instrumentation and techniques continue to improve image quality, contrast agents (CAs) can dramatically highlight anatomic and pathologic features of interest. Paramagnetic ions decrease the relaxation time of water protons. Thus, MR contrast agents are not directly imaged, but rather, indirectly affect the surrounding water molecules that in turn influence signal. Relaxation is also a function of molecular tumbling, the fast exchange between CA-coordinated water molecules (inner shell) and water in the bulk solvent (outer shells). If exchange among protons in the shells is rapid, they all exhibit similar relaxation behavior. The overall signal is then a weighted average of relaxation rates from each local proton environment, with the principal contribution from within the hydration sphere of the ion.
This article focuses on macromolecular MR contrast agents, including proteins, polypeptides, polysaccharides, poly (ethylene glycols) and dendrimers. However, it is instructive to highlight the basic chemical structures and main paramagnetic behavior of small molecule CAs currently in clinical use, as they are used as the paramagnetic component of macromolecular CAs.
Small Molecule MR Contrast Agents
Paramagnetic ions including Mn2+, Fe3+ and Gd3+ are potent class MRI contrast agents owing to their unpaired electrons, among which the lanthanide ion gadolinium with its 7 unpaired 4f electrons is by far the most commonly used agent. However, Gd ion is highly toxic in its free form, and thus a number of biocompatible chelating agents have been developed to render the metal ion nontoxic in its chelated form. These cages or chelates are designed with properties such as cavity size, geometry, donor character, and kinetic stability to have higher affinity for Gd3+ than for other metals commonly present in vivo such as Zn2+, Ca2+ and Cu2+, and thus the Gd chelates are highly stable in vivo.
One class of Gd complexes is macrocyclic chelates (), mostly derivatives of 1,4,7,10-tetraazacyclododecane (cyclen) (1
). The tetraacetic acid derivative complex with gadolinium, Gd-DOTA (gadoterate, Dotarem®) is formulated as its N
-methylglucamine salt, is highly water soluble, and thermodynamically stable. Two neutral macrocylic derivatives of 1,4,7-tricarboxymethyl-1,4,7,10-tetraazacyclododecane (DO3A) are gadoteridol (ProHance®) and gadobutrol, (Gadovist®). They are characterized by the replacement of one carboxylate with a hydroxyl donor group.
Chemical structures of cyclen (1,4,7,10-tetraazacyclododecane) and commercially available MRI contrast agents based on Gd chelates of cyclen derivatives.
Another class of such agents is acyclic chelates () which are comprised of derivatives of aminopolycarboxylic acids such as diethylenetriaminepentaacetic acid (DTPA). Gadolinium diethylenetriamine pentaacetic acid dimeglumine salt (Gd-DPTA), also known as Magnevist®, was approved for clinical use in adult patients in 1988 and has since become the most commonly used MR CA. The chelate has high thermodynamic stability and is water soluble (2
). Two diamide derivatives of DTPA were approved for human use: Gd-DTPA-BMA (gadodiamide, Omniscan®) and Gd-DTPA-BMEA (gadoversetamide, OptiMARK®). By reacting the dianhydride of DTPA with the corresponding amine (methyl amine or methoxyethyl amine, respectively), two carboxylates were replaced with two amide oxygen donors, resulting in a neutral charge on the chelate. These neutral agents remain highly water soluble and were developed in part to lower the osmolality of aqueous solutions (3
). However, in late 2006, the FDA issued a warning about exposure to gadolinium-containing CAs, increasing the risk for Nephrogenic Systemic Fibrosis in patients with severe kidney dysfunction (4
Chemical structures of acyclic commercially available MRI contrast agents based on Gd chelates.
While relaxivity is an important property, other factors also determine the efficacy of an agent in obtaining quality images, namely clearance rate. The amount of time it takes for a CA to be excreted, is dependent on a number of properties such as size, shape, surface charge and chemical makeup of the agent. With a molecular weight of 0.8 kDa, 90% of the injected dose of Gd-DPTA is cleared by renal filtration and vessel leakage in less than an hour (6
), hampering the ability to complete time-dependent imaging studies or obtain highly resolved images. However, the rapid clearance improves the safety profile, at least in subjects with normal renal function.
Macromolecular MR Contrast Agents
Staudinger introduced the macromolecular concept in 1920s, describing how monomers react with each other to form large (high molecular weights) molecules, that he called “macromolecules” (7
). This marked the beginning of macromolecular (polymer) science, and Staudinger was awarded the Nobel Prize in Chemistry for this work in 1953. The main types of natural and synthetic macromolecular or polymer architecture include linear, branched, graft (comb), multiarm (star), dendritic and crosslinked (or network). Most synthetic polymers are polydisperse, but a number of methods are also available for the synthesis of relatively monodisperse polymers. There are also a wide variety of functional polymers, both natural and synthetic, suitable for various biomedical applications including the delivery and targeting of drugs and contrast agents.
Macromolecular or polymeric metal-chelate complexes, also known as blood pool agents or macromolecular contrast media (MMCM), generally have a molecular weight greater than 30kDa and were originally designed for prolonged blood retention. Furthermore, they increase relaxivity due to slower molecular tumbling. The Solomon-Bloembergen-Morgan theory of paramagnetic relaxivity predicts that increasing the rotational correlation time of a paramagnetic ion with a relatively long electronic relaxation time, such as Gd3+
, will increase the ion’s relaxivity at clinically used field strengths (8
). The molecular dynamics of such paramagnetic ions dominate the dipole-dipole interactions between their unpaired electrons and the water protons in the inner shell (11
). Increased steric hindrance due to bulkier physical structure slows the rotation of larger molecules, increasing the rotational correlation time, τr
, and thus enhancing relaxivity and resulting in more enhancement per unit dose of the paramagnetic ion. Moreover, using a macromolecular structure potentially allows for the attachment of a large number of chelates and metal ions per agent further increasing enhancement. A lower local concentration of agent is thus required for satisfactory image acquisition.
In order to attach paramagnetic ions to macromolecular structures, a family of chelates known as bifunctional chelates () were developed, including 2-(4-isothiocyanatobenzyl)-6-methyl-diethylenetriamine pentaacetic acid (1B4M-DTPA), N
" pentaacetic acid (CHX-A-DTPA), N
"-pentaacetic acid (CHX-B-DTPA), and 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-N,N
"'-tetraacetic acid (p-SCN-Bz-DOTA) (13
). These allow the chelates to bind the metal ion as well as to a macromolecule.
Bifunctional chelates synthesized to attach to a ligand while also trapping a metal ion such as Gd3+. Diastereoisomers of CHX-DTPA chelates are shown.
A number of MR macromolecular contrast agents have developed over the last 30 years, ranging from protein to polymer to dendrimer-based molecules. These agents typically have diameters greater than 1–2nm as renal excretion slows enough for the agent to be effective. By 8nm, the liver begins to predominate as the clearance mechanism and completely dominates by 10–12nm. Biodistribution differs significantly with long retention times and limited extravasation. While MR agents such as liposomes and gold and iron nanoparticles are beyond the scope of this article, Gd-chelate based structures are discussed herein.