Oxidative conversion of a Eu(II)-based T₁ agent to a Eu(III)-based paraCEST agent can be detected in vivo by MRI

Abstract

In this work, we demonstrate that Eu(II) complex of DOTA-tetra(glycinate) has a higher reduction potential than most Eu(II) chelates reported so far. The reduced Eu(II) form acts as an efficient water proton T₁ relaxation reagent while the Eu(III) form acts as a water-based CEST agent. The complex has extremely fast water exchange rate. Oxidation to the corresponding Eu(III) complex yields a well-defined signal from the paraCEST agent. The time course of oxidation was studied in vitro and in vivo by T₁-weighted and CEST imaging.

Graphical Abstract

A Eu(II) complex of DOTA-tetra(glycinate) has far less negative redox potential than most Eu(II) chelates reported so far. The reduced Eu(II) form acts as an efficient water proton T₁ relaxation reagent while the Eu(III) form acts as a water-based CEST agent. The time course of oxidation was studied in vitro and in vivo by T₁-weighted and CEST imaging.

Gadolinium complexes are commonly used as contrast agents in magnetic resonance imaging (MRI). They generate image contrast by shortening the longitudinal (T₁) relaxation time of bulk water protons. The efficiency of a T₁- agent is defined by r₁ relaxivity, which is dependent on several parameters including the metal bound water exchange rate, rotational correlation time of the complex and the electronic relaxation time of the metal ion.^[1] An alternative to the Gd³⁺-based contrast agents is the isoelectronic Eu²⁺ ion.^[2] Both ions have a 4f⁷ electron configuration and a symmetric ⁸S_7/2 ground state but Eu²⁺ complexes in general display much faster water exchange rates and faster electronic relaxation times.^[2] Analogous complexes of Eu²⁺ and Gd³⁺ can produce similar relaxivity values at lower fields while at higher fields the Eu²⁺ complexes tend to be more efficient.^[3]

The Eu²⁺ aqua ion is extremely sensitive to oxidation as demonstrated by its strongly negative reduction potential (−585 mV vs Ag⁺/AgCl). Eu²⁺ poly(amino carboxylate) chelates usually have lower reduction potential although some Eu²⁺-cryptates have been reported to be more stable towards oxidation.^[4,5] Eu²⁺ complexes have been proposed as redox sensitive T₁ agents,^[3,5,6] because oxidation of Eu²⁺ leads to the formation of weakly paramagnetic Eu³⁺, which has little impact on water proton T₁. The Eu³⁺ ion however, generates a moderately strong magnetic dipolar field that produces large hyperfine shifts of NMR signals of nearby ligand protons. While Eu³⁺ complexes are very poor T₁-shortening agents, Eu³⁺ DOTA tetra(amides) (Figure 1) belong to a conceptually different class of MRI contrast agents, known as paraCEST agents that alter image contrast by transferring selectively saturated spins from a highly shifted small pool of proton spins (metal bound water) to the bulk water pool.^[7] Chemical exchange saturation transfer (CEST) occurs when the proton exchange rate between the two pools (k_ex) is in the slow-to-intermediate exchange regime (k_ex ≤ Δω where Δω is the chemical shift difference between the two pools). A redox responsive liposomal Eu²⁺/Eu³⁺ system was recently reported that showed T₁ shortening effect and lipoCEST effect (Δω ≈ 1ppm) originating from the exchange between the water protons inside the liposomes and the bulk water protons not associated with the liposomes. Upon oxidation, the T₁ enhancement disappeared while the lipoCEST remained unaffected. The CEST effect in this system is not due to the formation of a Eu³⁺ complex.^[8]

Figure 1.

Structure of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 1 and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tetra(glycine amide) 2.

Eu²⁺ complexes have several orders of magnitude faster water exchange rates in comparison to the corresponding Eu³⁺ complexes and with a suitable ligand system, may offer a unique opportunity in the design of redox responsive MR agent that shortens T₁ in the reduced state and produces a CEST signal in the oxidized state. In the present work we show that Eu²⁺2 is an efficient T₁ shortening agent because of the rapid water exchange of the Eu²⁺ bound water, but upon oxidation it turns into the well-known paraCEST agent, Eu³⁺2, which has slow water exchange kinetics. Merbach suggested in 2003 that the redox stability of Eu²⁺1 could be increased by substituting nitrogen containing donor groups for the carboxylate side-arms.^[3,6] Here, we also show that Eu²⁺2 indeed has significantly improved redox stability compared to Eu²⁺1.

The CEST effect can be expressed as a decrease in total bulk water signal intensity, and assuming complete and instantaneous saturation of the bound water peak, the net magnetization of water protons at steady-state is given by the following equation:

$$\mathit{CEST}\mathit{effect}(\%)=1-\left(\frac{M_s}{M_0}\right)=100{\left(1+\frac{cq{T}_1}{111{\tau }_m}\right)}^{-1}$$ (1)

where c is the concentration of the agent, q is the number of protons per agent, 111 represents the molar concentration of bulk water protons, T₁ is the longitudinal (spin–lattice) relaxation time of bulk water, and τ_M is the lifetime of the exchanging proton (τ_M = 1/k_ex).^[7] Thus, the magnitude of the CEST effect is dependent on both the agent concentration and the bulk water T₁. Obviously, as Eu²⁺2 are oxidized, the T₁ shortening effect of Eu²⁺ will diminish while the paraCEST agent concentration will increase over the course of the reaction. To study the dependence of CEST on T₁, we designed a model experiment in which the paraCEST agent Eu³⁺2 were mixed with varying concentration of the T₁ shortening agent Gd³⁺1 (Table S1). Figure 2 shows that the proton relaxation rate (R₁ = 1/T₁) of bulk water protons increases with increasing [Gd³⁺1] while the CEST signal from the paraCEST agent diminishes. At the two extremes of [Gd³⁺1], the images are dominated by either CEST (when [Gd³⁺1] = 0) or T₁ (when [Gd³⁺1] = 4 mM) but there is a range of Gd³⁺ concentrations (samples 4, 5 and 6) where both CEST and T₁ enhancement contribute to the signal. The CEST signal was <10% when R₁ = 5 s⁻¹. From the fitting of the T₁ and CEST intensities to equation (1), a bound water residence lifetime (τ_M) of 410 ms was obtained for Eu³⁺2 at 19°C, in agreement with the τ_M value determined by other methods.^[9] This same phenomenon, the sensitivity of CEST to water proton T₁, formed the basis of a recently reported redox-sensitive paraCEST agent.

Figure 2.

(Top) Correlation between relaxation rates (circles) and the CEST effect (squares) in a mixture of Gd³⁺1 and Eu³⁺2. Each circle in the image represents a separate sample. [Eu³⁺2] in each sample was 10 mM while [Gd³⁺1] ranged from 0 to 4 mM. Vial 9 contained 8 mM Gd³⁺1 alone while vial w contained only water. (Bottom) Plots of CEST (1-(M_s/M₀)) and R₁ versus [Gd³⁺1]. Imaging parameters: B₀=9.4 T, 20 °C; T₁w: GEMS sequence, TR= 9.9 ms, TE= 5.0 ms; CEST: FSEMS sequence, sat time = 3 s, sat power = 10 μT, sat frq = 54 (on),-54 ppm (off-resonance).

Eu²⁺ complexes of 1 and 2 were conveniently prepared by directly reacting the ligands with commercially available EuCl₂ under oxygen free conditions.^[10] The relaxivity of Eu²⁺2 was measured as 3.2 mM⁻¹ s⁻¹ at 9.4T and 1 T (Figures S1 and S2). The Eu²⁺ bound water exchange rate as estimated by fits of variable temperature ¹⁷O NMR water linewidth data to theory was k_ex = 0.21 × 10⁹ s⁻¹ for Eu²⁺2 and k_ex = 0.63 × 10⁹ s⁻¹ for Eu²⁺1 in 20% dioxane − 80% water solutions (Figure S3, S4 and Table S2–S4), which are in the range of previously reported values.^[3,6,11,12] It is worth noting that the water exchange for Eu²⁺2 is nearly the same as that of Eu²⁺1. This indicates that the glycinate amide side-chains in Eu²⁺2 do not affect the water exchange rate in comparison to the corresponding Eu³⁺ complexes where the difference between carboxylate and amide donor ligands is typically 3 orders of magnitude.^[1,2] Unlike Gd³⁺ complexes, the r₁ value of Eu²⁺2 did not decrease significantly at high field, in agreement with previously published data for other Eu²⁺ complexes. The redox stability of Eu²⁺2 was studied by cyclic voltammetry. The reduction potential measured for the Eu²⁺2/Eu³⁺2 redox couple was found to be −226 mV vs. Ag⁺/AgCl electrode (Figure S5), which is far less negative than that of Eu²⁺1 (−1135 mV), or than the Eu²⁺ aqua ion (−585 mV against Ag⁺/AgCl electrode).^[3] The rates of conversion of Eu²⁺2 and Eu²⁺1 to their respective Eu³⁺ complexes were also investigated by NMR by measuring the decay of the relaxation rate of the bulk water in a sealed NMR tube under N₂ atmosphere at 9.4T (Figures S6 and S7). The measured half lives (t_1/2), 19 and 7 days respectively, also show that Eu²⁺2 is the most stable cyclen-based Eu²⁺ complex reported so far. Similar stabilizing effect of the charge neutral amide group was reported for Eu²⁺ complexes of 1,10-diaza-18-crown-6 derivatives in which picolinamide pendant arms were substituted for picolinate groups.^[13]

Next, a fresh solution of Eu²⁺2 was prepared in a 6 mm tall vessel and exposed to air while both T₁ and CEST were measured as a function of time by imaging a slice 4 mm below the surface of the sample (Figure 3). As shown, the bulk water R₁ decreased steadily for about 100 minutes in this slice reflecting oxidation of Eu²⁺ to Eu³⁺. The CEST signal increased following a sigmoid curve with an inflection point at around 100 min reaching 10% at around 6 s⁻¹ bulk water relaxation rate in agreement with the results shown in Figure 2. These data reveal that relaxation of bulk water protons limits the intensity of the CEST signal and at ~80 min, the T₁ relaxation is slow enough for CEST to become efficient. As expected, the rate of oxidation depends heavily on the slice selected. For example, the T₁w enhancement in a slice just below the surface disappeared just after 10 minutes (Figure S8).

Figure 3.

Plots of relaxation rate (R₁) (circles) and CEST (squares) for a solution initially containing 10 mM Eu²⁺2 versus time. The data were collected from images in a single slice 4 mm away from the surface. Imaging parameters: B₀=9.4 T, 20 °C, T₁w: FSEMS sequence, TR = 2 s, TE = 3.0 ms; CEST: FSEMS sequence, sat time = 3 s, sat power = 10 μT, sat frq = 54 (on-resonance), -54 ppm (off-resonance).

To demonstrate the feasibility of using Eu²⁺2 as redox sensitive probe we studied its reaction with H₂O₂. A closed phantom containing Eu²⁺2 solution (1 mL, 10 mM) was constructed and a small volume (20 μL) of hydrogen peroxide solution (3%) was injected into the container. T₁w and CEST images were recorded consecutively over a period of 1 h. As anticipated, the complex reacted rapidly with H₂O₂ and the mixing and diffusion of H₂O₂ in the solution could sequentially be observed by both T₁w and CEST imaging. These images also demonstrate that the complex is stable in both oxidation states and throughout the process of oxidation (Figure 4 and S9, S10).

Figure 4.

Sequential T₁w (top) and CEST (bottom) images of a phantom containing Eu²⁺2 (1 mL, 10 mM, left) and H₂O (right) after the injection of H₂O₂ (3%, 20 μL). Imaging parameters: B₀=9.4 T, 20°C T₁w: FSEMS sequence, TR = 2 s, TE = 5 ms; PARACEST: FSEMS sequence, sat time = 3 s, sat power = 10 μT, sat frq = 54 (on-resonance),-54 ppm (off-resonance).

Encouraged by the enhanced stability of Eu²⁺2 we tested the agent in vivo as well. The complex was injected into the thigh muscle of healthy female mice at a dose of 0.05 mmol/kg. Oxidation of the agent at the injection site could be observed by both T₁w and CEST imaging (Figure 5). Given that diffusion of small molecules away from the injection site and into the vascular bed occurs relatively slowly, the relatively rapid decrease in water proton enhancement seen in T₁w images at the injection site over ~15 min presumably reflects oxidation of Eu²⁺ to Eu³⁺. To validate this assumption, after T₁w enhancement of the signal vanished, CEST images were collected to verify that Eu³⁺2 was indeed present. The CEST signal detected near the injection site (Figure 5e) remained evident over a period of ~20 min before disappearing (Figure S11).

Figure 5.

T₁w and CEST imaging of an intramuscular injection of Eu²⁺2 (10 mM, 100 μl) into the thigh muscle of a healthy female C57/blk6 mouse at B₀=9.4 T. T₁w images at a) pre-injection, b) 5 min, c) 12 min, d) 16 min and e) the CEST image at 17 min. Selected imaging parameters: T₁w: ge3D sequence, TR=3.6 ms, TE=1.8 ms. T₁w images have been normalized to muscle tissue; PARACEST: FSEMS sequence, sat time = 3 s, sat power = 10 μT, sat frq = 42 (on-resonance),-42 (off-resonance) ppm.

Since free Eu³⁺ does not produce a CEST effect, the detection of the agent by CEST imaging after the T₁w enhancement indicates that the complex remained intact in vivo throughout the oxidation process. All of the mice recovered after imaging and no evidence of toxicity was apparent after injection of Eu²⁺2.

In conclusion, we have shown that ligand 2 forms a complex with Eu²⁺ that is surprisingly stable to oxidation. The Eu²⁺-bound water exchange rate for this complex (k_ex = 0.21 × 10⁹ s⁻¹) was found to be extremely fast, indicating that the amide sidearms do not have a significant decelerating effect on the water exchange. The agent has different contrast enhancing properties depending on the oxidation state of the metal. In its divalent form it is an efficient T₁ shortening agent with an r₁ relaxivity comparable to Gd-based contrast agents. Oxidation converts it into Eu³⁺2, which is a commonly used paramagnetic chemical exchange saturation (paraCEST) agent. The oxidation of Eu²⁺2 by air or H₂O₂ could be followed by both T₁w and CEST imaging. The improved oxidative stability of Eu2+2 When injected intramuscularly into healthy mice the complex generated strong T₁ enhanchement that gradually diminished over several minutes after which strong CEST effect could be observed at the injection site. This complex could serve as a design platform for a novel class of redox sensitive bimodal MR contrast agents in which the redox potential of the Eu²⁺ may be fine-tuned by the nature of the peripheral amide groups.