Hydrochalarones: A Novel Endohedral Metallofullerene Platform for Enhancing Magnetic Resonance Imaging Contrast

Abstract

Targeted imaging requires contrast agents that remain in the vasculature for extended periods of time. A new contrast agent is described in which gadolinium is encapsulated within an extremely stable carbon sphere, thus allowing for safe extended residence. Water solubility and small particle size is achieved with novel fullerene chemistry, attaching multiple oligoethylene glycol groups through nitrogen chemistry. These new compounds can be used to visualize tissue architecture in vivo with standard MRI techniques.

Discoveries in molecular biology have produced an explosive growth in efforts to identify new targets for diagnostic medicine. However, imaging the distribution of validated targets in patients has lagged for want of safe, high-resolution imaging tools. Some nuclear medicine agents are targeted, but the relatively low resolution of PET/SPECT requires that these methods be combined with CT scans for interpretation, and there is concern about the long-term effects of exposure to ionizing radiation.¹ Magnetic resonance imaging (MRI) is widely available, highly translatable, and can provide high contrast, especially for the study of soft tissue. However, recent concerns over the role of current Gd-based contrast agents in the development of nephrogenic systemic fibrosis (NSF) in patients with impaired renal function^2,3 highlight the need for safer imaging agents with enhanced imaging capabilities. In this report, we describe a new technology for improving MRI contrast based on endohedral metallofullerenes containing Gd. Surface modification of the fullerene provides much higher spin coupling with water protons than other forms of Gd despite separation by the fullerene cage. These nanomaterials are remarkably stable and hence are unlikely to release the Gd. These new nanomaterials injected intravenously demonstrate enhanced contrast in a mouse MRI, with circulation of at least 30 min. This technology could serve as a platform for developing agents that can specifically target molecular entities uniquely expressed on certain cells, tissues, or organs, thus providing caregivers improved ways to diagnose, treat, and monitor progression of a wide range of diseases.

The fullerene-based nanomaterials described here may provide a unique platform for developing such technologies based on their enhanced relaxivity, stability, and ability to be derivatized with molecular targeting moieties. Endohedral metallofullerenes containing one Gd atom,^4,5 derivatized by polyhydroxylation or through addition of carboxyl groups to improve water solubility, exhibit high relaxivity, though the relaxivity has been attributed primarily to the formation of aggregates.^6,7 This problem was overcome through chemistry that adds groups onto the fullerene cage that optimize magnetic coupling between the unpaired electrons on the enclosed paramagnetic Gd and water protons outside the cage. The cage is extremely stable; extreme oxidizing conditions are required to release gadolinium from the cage.

The first trimetallic nitride MRI contrast agent, based on Gd₃N@C₈₀, was hydroxylated and PEGylated with long-chain glycols.⁸ In the present work, we have synthesized⁹ a series of novel derivatives of Gd₃N@C₈₀ termed Hydrochalarones (derived from χαλαρω = relax). These Hydrochalarones are not hydroxylated but are made water soluble by addition of a series of glycol methyl ethers, ranging from monoethylene glycol to hexaethylene glycol (Figure 1A). Importantly, the polar ethylene glycols are extremely close to the fullerene cage which increases its water solubility. The paramagnetic Gd atoms preclude using standard NMR to confirm the structure of these nanomaterials, but the number of attached groups can be inferred from thermogravimetric (TGA) analysis, where oxidation of the side chains takes place at a lower temperature than the cage. The TGA measurements on Gd₃N@C₈₀ and Hydrochalarones revealed the number of attachments on the cage to be 20–22, 12–14, and 10–12 for Hydrochalarone-1, -3, and -6, respectively. The connectivity between fullerene and glycol is not clear but likely includes RNOH and isoxazoline-like rings as inferred from their IR stretches (Figure 1B).

Figure 1.

(A) General structure of Hydrochalarones is Gd₃N@C₈₀-R_x, where R = −[N(OH)(CH₂CH₂O)_nCH₃]_x where n = 1,3,6 and x is 10–22. (B) Infrared spectra of Hydrochalarone-6.

It is known that a very stable species Gd₃N@C₈₀ is formed by combining two unstable species, Gd₃N and C₈₀. The Hydrochalarone derivatives are even more stable. For example, TGA shows Gd₃N@C₈₀ begins to oxidize at 180 °C and disintegrates very rapidly thereafter. By contrast, the Hydrochalarone cages start to decompose at ∼500 °C. Apparently the addition of polyethylene glycol groups stabilizes the carbon cage, presumably by relieving stress on the C=C bonds. Similarly, even heating in concentrated nitric acid at 80 °C for 4 h does not release the Gd atoms from the cage. Hydrochalarones are stable at room temperature for at least 6 months, monitored by relaxivity.

The particle size of these nanomaterials was measured using DLS and found to be 10–15 nm, which is small enough to circulate through capillaries. Measurements of T₁ relaxivity, r₁, the standard metric of contrast enhancement, were performed on these nanomaterials in water using a 20 MHz Maran Ultra relaxometer (Oxford Instruments). The optimal member of this series, Hydrochalarone-6, has r₁ of 205 mM⁻¹ s⁻¹ (68 mM⁻¹ s⁻¹ Gd, compared with 3.8 mM⁻¹ s⁻¹ for Magnevist, a commercial product containing Gd DTPA). Interestingly, the ratio of T₂ relaxivity to T₁ relaxivity, r₂/r₁, is comparable to those of the small Gd chelates, which is a critical factor in controlling signal intensity during T₁-weighted MRI.

Chelate-based MRI contrast agents, with one or more coordinated water molecules that exchange with bulk water molecules, exhibit a relaxivity described by the Solomon–Bloembergen–Morgan (SBM) equation.¹⁰ In Hydrochalarone, the Gd atoms are entrapped in the cage and there is no possibility of water being coordinated to the paramagnetic metal centers. Thus, a different mechanism of spin transfer between Gd and water must be at work.

Previous work on Gd@C₆₀(OH)_x and Gd@C₆₀-[C(COOH_yNa_1−y)₂]₁₀ implicated proton exchange from bulk water with hydroxyl and protonated malonate groups as integral to magnetic coupling.⁴ Application of this hypothesis to Hydrochalarones can be tested by looking for −OH/−OD exchange in the FTIR spectrum. During deuterium exchange experiments no −OD stretches were observed, indicating that proton exchange cannot account for the relaxivity observed.

The X-ray crystal structure shows that the Gd₃N@C₈₀ cage is slightly distorted by the entrapped Gd₃N species and the Gd atoms are closer to the carbon atoms of the cage.¹¹ The unpaired f-orbital electrons of the Gd atoms may interact with electrons in the molecular orbitals on the six-carbon rings near the distortions. Water forms weak hydrogen bonds on benzene rings.¹² Gel electrophoresis shows that Hydrochalarones bear a negative charge that could enhance hydrogen bonding. Unpaired f-orbital electrons of Gd may couple their spins through the electrons on the carbon molecular orbitals to water protons that are hydrogen-bonded to the cage surface. This represents another example of “spin leakage”^13,14 and supports the hypothesis that metallofullerenes behave as “superatoms.”¹⁵ Hydrogen-bonded water molecules may act like inner-sphere water that rapidly exchange with many surrounding water molecules. Different length polyethylene glycol attachments may alter relaxivity by changing water diffusion rates through the hydration shells of the polyethylene glycol moieties.

In vivo MRI studies were performed at 4.7 T using a small-animal scanner interfaced to a Varian INOVA console. Hydochalarone-6 was administered intravenously to a BALB/c mouse at a dose of 0.01 mmol/kg. (A typical clinical dose of commercial Gd-based MRI contrast agent is 0.1 mmol/kg.) Figure 2 shows a coronal slice from a T₁-weighted whole-body image of a mouse before and 30 min after Hydrochalarone administration. The persistent image enhancement of the aorta (arrow) indicates Hydrochalarone-6 remains in the vasculature, consistent with the observation that polyethylene glycol modifications increase the circulation time of proteins.¹⁶

Figure 2.

Coronal slice from a whole-body MRI of a mouse before (left) and 30 min after (right) administration of Hydrochalarone-6. The unidentified bright spot in both the pre- and postcontrast images is likely an artifact.

In summary, Hydrochalarones are stable, appear well-tolerated in vivo, provide excellent MRI enhancement, and have “stealth” properties, meaning that rapid clearance mechanisms seem not to be triggered. These are desirable properties for the development of Hydrochalarones as imaging agent platforms upon which can be attached a broad range of targeting species for improved clinical diagnosis and management of a number of diseases.