Near Infrared-Fluorescent and Magnetic Resonance Imaging Molecular Probe with High T1 Relaxivity for In Vivo Multimodal Imaging

Abstract

A new gadolinium chelating NIR fluorescent molecular probe increases T₁ relaxivity of water protons, facilitating combined optical and magnetic resonance imaging.

Integration of optical and magnetic resonance imaging (MRI) methods in one setting is appealing due to the complementary nature of MRI’s high spatial and temporal resolution and the high molecular sensitivity of optical imaging.¹ In particular, deep tissue penetration of near-infrared (NIR) photons and minimal autofluorescence in this wavelength range² favors the assessment of molecular events in thick tissue by optical in vivo imaging.³ A major challenge in constructing dual MRI/optical probes is matching the relatively low contrast agent’s detection sensitivity (micromolar) by MRI with the single molecule detection capability of fluorescence imaging. Herein, we report a new molecular design consisting of a Gd³⁺ chelating NIR carbocyanine dye (LS-479-Gd³⁺; Fig. 1) to bridge the sensitivity gap between the two imaging modalities. We envisioned that MRI sensitivity could be improved by decreasing the rotational diffusion of the molecular probe, which could be achieved by anchoring the probe to a macromolecule such as a protein. With this dual Gd³⁺-fluorophore probe, we were able to obtain high T₁ weighted MRI and fluorescence imaging contrast in small animals.

Fig. 1.

Structures of LS479-Gd3+ and Magnevist®

MRI produces high spatial resolution images of soft tissue by measuring the relaxation rate of water protons⁴. With their high magnetic moment, paramagnetic lanthanide ions increase the relaxation rate of proximal protons and provide contrast. High relaxivity translates into improved detection sensitivity at relatively lower contrast agent concentration than is traditionally used for MRI.⁴ Gd³⁺ is the most commonly used MRI contrast agent due to its optimal electronic characteristics.⁵ The high toxicity of free Gd³⁺ requires its incorporation in a chelating matrix,⁶ hence a variety of gadolinium chelates have been developed, either as nonspecific extracellular agents or as targeted agents optimized for specific medical applications.⁷

In general, total relaxivity of an MRI contrast agent depends on a number of factors, including rate of water exchange and its rotational diffusion.⁸ Total relaxivity is a combination of inner and outer sphere relaxivities. The inner sphere relaxivity is given by the following equation:⁴

$$1/T_1=(P_m\times q)/(T_{1m}+{\tau }_m)$$

where P_m is the mole fraction of the metal ion, q is the number of water molecules bound per metal ion, T_1m is the relaxation time of the coordinated water molecule(s), and τ_m is the residence lifetime of the coordinated water.

For metals such as Gd³⁺ with a long electronic spin relaxation time,⁴ the important factor in increasing T₁ of water is to limit rotational diffusion of the metal (i.e. decreasing T_1m). An effective method to limit rotational diffusion of a probe is binding the contrast agent to a macromolecule with a slow rotational time, such as a protein.⁹ For example, the relaxivity of the clinically used MRI contrast reagent Magnevist^® (Gd³⁺-DTPA) with negligible albumin binding has a T₁ relaxivity of 4–5 mM⁻¹ sec⁻¹, ¹⁰ while a modified version of Gd³⁺-DTPA complex (Vasovist^®, ¹⁰ with high albumin binding affinity, K~ 11,000 M⁻¹, see SI for binding constant measurement) has a much higher T₁ relaxivity of ~50 mM⁻¹ sec⁻¹ in human plasma¹⁰ due to a 60–100 fold decrease in rotational diffusion.¹¹ While covalent binding is possible,¹² noncovalent interactions with protein, as in the case of Vasovist^®, is attractive to facilitate the eventual clearance of the probe via normal excretory pathways.⁴

We demonstrated previously that NIR polymethine dyes bind noncovalently to serum albumin and exhibit increased quantum yield and fluorescence lifetime due to stabilization of the fluorophore when enclosed in the hydrophobic pockets of albumin.¹³ In this study, we hypothesized that opsonization of a NIR dye-Gd³⁺ complex possessing a metal chelating group electronically coupled to the fluorophore of the fluorophore could further induce rigidification of the metal chelating group, dramatically reducing the molecular tumbling of the metal-chelate system and enhancing T₁ relaxivity. This disposition will synergistically enhance the fluorescence signal as well as the T₁ signal that is useful for functional dual MRI-optical imaging.

We selected LS479, a NIR polymethine dye electronically coupled to a DTPA chelating group (Fig. 1). The compound was synthesized as previously described.¹⁴ The sensitivity of LS479-Gd³⁺ to serum albumin (we used bovine serum albumin as a model) was compared to Magnevist^®, a DTPA-based clinical contrast agent for MRI.

Previously, we had shown that complexation of LS479 to certain metals induces spectral changes (absorption and emission).¹⁴ On this basis, we evaluated the use of spectroscopic method to track the complexation of Gd³⁺ to LS479. As expected, titration of LS479 with Gd³⁺ resulted in concentration dependent fluorescence (Fig. 2) and absorption (Fig. S1) spectral changes. Concentration induced changes in the absorption spectra of LS479 showed clear isosbestic points at ~715 nm and ~755 nm. We did not observe further changes after saturation of LS479 with Gd³⁺. At saturation (1:1 dye to metal ratio), LS479 displayed ~700% enhancement of fluorescence. The enhancement was attributed to the suppression of excited state electron transfer, as previously described.¹⁴ Photophysical properties of LS479 and LS479-Gd³⁺ in 4% BSA are tabulated in Table S1.

Fig. 2.

(A) Titration emission spectra of LS479 with GdCl3 in water (ex. 675 nm), arrow indicates the direction of increasing Gd3+ concentration from 0 to 1.2 metal/dye ratio; (B) fluorescence intensities at emission maxima (770 nm) as a function of metal/dye ratio.

Free Gd³⁺ is toxic, often leading to fatal nephrogenic systemic fibrosis (NSF),¹⁵ especially in patients with renal failure. For that reason, Gd³⁺ must remain trapped in a chelating molecule and safely excreted from the body. Having demonstrated the complexation of Gd³⁺ to LS479, we further established that LS479-Gd³⁺ binds to albumin and the dye-metal complex remains intact, making it a viable contrast agent for MRI applications.

First, we evaluated BSA binding affinity of LS479-Gd³⁺, (see ESI for the experimental method). The binding constants of LS479 (~48,000 M⁻¹) and LS479-Gd³⁺ (~35,000 M⁻¹) were determined by measuring the slope in a concentration vs. tryptophan emission intensity plot (Fig. S2). The large and similar binding constants of the two compounds indicate that chelation of Gd³⁺ by LS479 does not significantly alter the binding properties of the dye to BSA. Second, measurement of the fluorescence lifetime of LS479-Gd³⁺ in albumin showed a longer decay compared to albumin-free aqueous solution (Fig. S4), with an average lifetime increase of ~2.6 fold (Table S2). This change is typical of opsonized NIR cyanine dyes^{13, 16} and confirms that the dye is bound to the protein. The spectral profile and fluorescence lifetime of LS479-Gd³⁺ were stable over a long period, evidencing the stability of the complex in the bound state. Finally, a small but noticeable change in the absorption and emission spectra of LS479-Gd³⁺ in albumin compared to the metal-free LS479 in albumin was observed (Fig. S3), indicating chelation of the metal.

After demonstrating the stability of LS479-Gd³⁺ in albumin, we next determined the T₁ relaxivity of LS479-Gd³⁺ and compared it to that of Magnevist^® in 4% BSA and water to mimic natural concentrations of albumin in blood. Relaxivities of the samples were determined from the slopes of a linear plot of 1/ T₁ vs. concentration of contrast agent (Fig. 3). The relaxivity of LS479-Gd³⁺ in water (~15 mM⁻¹ sec⁻¹) was higher than that of Magnevist^® (~3.9 mM⁻¹ sec⁻¹), probably due to the presence of two coordinated water molecules characteristic of Gd³⁺-DTPA derivatives¹⁷ used in LS479-Gd³⁺ compared to the single inner sphere water of Gd³⁺-DTPA of Magnevist^®. In this configuration, the nine-coordinate Gd³⁺ has coordination number of seven in LS479-Gd³⁺ and eight in Magnevist^®, as shown in Fig. 1. In the presence of albumin, the T1 relaxivity difference was further magnified. LS479-Gd³⁺ bound to albumin showed a relaxivity of ~34 mM⁻¹ sec⁻¹, a ~2.5 fold enhancement over its relaxivity in water while Magnevist^® increased by a small margin of ~1.2, which is close to the published enhancement of 1.5 in human serum albumin.¹⁸

Fig. 3.

T₁ relaxivities: LS479-Gd³⁺ in albumin (34.05 mM⁻¹ sec⁻¹, R²=0.999), LS479-Gd³⁺ in water (15.02 mM⁻¹ sec⁻¹, R²=0.990), Magnevist^® in albumin (4.93 mM⁻¹ sec⁻¹, R²=0.999), Magnevist^® in water (3.87 mM⁻¹ sec⁻¹, R²=0.999). Field strength-23 MHz.

Encouraged by the remarkable enhancement of LS479-Gd³⁺ T₁ relaxivity in the presence of BSA, we proceeded to evaluate the potential of using this compound as a multimodal contrast agent for MRI and optical imaging in mice. 3D FLASH MR images were acquired after intravenous injection of 0.025 mmol/kg in a mouse. The T₁ weighted images showed substantially higher T₁ signal in the jugular, subclavian, and thoracid veins as well as the liver, aorta and surrounding tissues at 2 min and 4 min following injection of the compound (Fig. 4).

Fig. 4.

T₁ weighted MR images of a mouse before (A), two minutes (B) and four minutes (C) acquired post injection of LS479-Gd³⁺. (See Table S3 for SNR values and Fig. S5 for longitudinal study.)

We observed an increase in the signal-to-noise ratio (SNR) of ~235% and 100% for the liver and heart septum, respectively, and negligible change for muscle tissue after 2 min (see Table S3 for the raw data). The SNR for the heart septum and liver decreased rapidly after injection (Fig. S5), apparently due to rapid extravasation of LS479-Gd³⁺.

Similarly, noninvasive optical imaging showed strong fluorescence signal throughout the body at 40 min post injection, with intense fluorescence in the spine (Fig. 5A). After 72 h, the fluorescence reduced rapidly and the residual agent remaining in the body (less than 4%) was primarily localized in the neck and spine regions (Fig. 5B).

Fig. 5.

Fluorescence intensity image of the mouse 40 min (A) and 72 h (B) post injection demonstrate accumulation in the neck and spine. Whole body fluorescence lifetime images 40 min (C) and 72 h after injection (D). (See Table S4 for lifetime values.

Previous studies have shown that the fluorescence lifetime of similar dyes is sensitive to the nature of their biological environment.¹⁶ In high polarity urine, the lifetime is expected to be lower than in blood, where the dye is bound to hydrophobic albumin binding sites.^{13, 19} As shown in Fig. 5C, the long lifetime of about 1 ns indicates that the dye is in circulation and the pockets of shorter lifetimes (~0.8 ns) in the vicinity of the liver and bladder suggest the extravasation of some of the agent to tissue. After 72 h, the shorter lifetime (blue) map in vicinity of the kidneys indicates that some of the compounds were cleared by the kidney. Published reports have shown that other hydrophilic NIR fluorescent dyes have similar renal clearance characteristics.¹⁹ Although the albumin-bound cyanine dyes such as ICG are expected to be excreted predominantly by hepatobiliary pathway, the unbound LS479-Gd³⁺ is a hydrophilic molecule that can be excreted by the kidneys. Interestingly, despite the relatively large amounts of LS479-Gd³⁺ used in this study to compensate for the low contrast agent detection sensitivity of MRI, the mice remained in apparent good health for more than 2 months after injection.

In conclusion, we have developed a new NIR optical-MRI imaging probe, LS479-Gd³⁺, that utilizes the fluorophore itself as a binding scaffold to serum albumin. We showed that upon binding to albumin, the fluorophore-metal chelate system significantly enhanced the T₁ relaxivity attributable to the induced rigidification of the chelating group. Preliminary in vivo results indicate the potential use of the imaging agent for dual optical-MRI studies. This study illustrates a synergistic approach to increase the fluorescence intensity and T₁ relaxivity for potential dual optical and MRI imaging studies. Specifically, the concept can be used to develop new molecular constructs for enhanced dual MRI/optical imaging.