A cell-permeable gadolinium contrast agent for magnetic resonance imaging of copper in a Menkes disease model

Abstract

We present the synthesis and characterization of octaarginine-conjugated Copper-Gad-2 (Arg₈CG2), a new copper-responsive magnetic resonance imaging (MRI) contrast agent that combines a Gd³⁺-DO3A scaffold with a thioether-rich receptor for copper recognition. The inclusion of a polyarginine appendage leads to a marked increase in cellular uptake compared to previously reported MRI-based copper sensors of the CG family. Arg₈CG2 exhibits a 220% increase in relaxivity (r₁ = 3.9 to 12.5 mM^-1 s^-1) upon 1 : 1 binding with Cu⁺, with a highly selective response to Cu⁺ over other biologically relevant metal ions. Moreover, Arg₈CG2 accumulates in cells at nine-fold greater concentrations than the parent CG2 lacking the polyarginine functionality and is retained well in the cell after washing. In cellulo relaxivity measurements and T₁-weighted phantom images using a Menkes disease model cell line demonstrate the utility of Arg₈CG2 to report on biological perturbations of exchangeable copper pools.

Fig. 1

Interaction of Arg₈CG2 with Cu⁺. (a) Plot of r₁ relaxivity versus added Cu⁺ for a 0.2 mM solution of Arg₈CG2 in PBS (pH 7.4), at 37 °C at a proton Larmor frequency of 60 MHz. (b) Normalized absorbance response of 0.2 mM Arg₈CG2 to buffered Cu⁺ solutions for determination of the apparent K_d value. Spectra were acquired in PBS (pH 7.4) at 25 °C. The black trace represents the best fit (K_d = 8.3 × 10⁻¹⁵ M, R² = 0.986).

Fig. 2

Relaxivity responses of Arg₈CG2 to various metal ions. White bars represent the addition of the appropriate metal ion (10 mM for Na⁺; 2 mM for K⁺, Mg²⁺, and Ca²⁺; 0.2 mM for Fe²⁺, Fe³⁺ and Cu²⁺) to 0.2 mM Arg₈CG2. Response to Zn²⁺ was measured both at 0.2 mM Zn²⁺ (Zn²⁺ 1 ×) and 2 mM Zn²⁺ (Zn²⁺ 10 ×). Black bars represent the subsequent addition of 0.2 mM Cu⁺ to the contrast agent solution. Relaxivity measurements were acquired at 37 °C in PBS (pH 7.4) at a proton Larmor frequency of 60 MHz.

Fig. 3

Cellular uptake of complexes in HEK 293T cells. (a) CG2 (black) and Arg₈CG2 (gray) in HEK 293T cells following 1 h incubation at various dosing concentrations. After incubation, cells were rinsed with PBS, lysed with RIPA buffer, dissolved in nitric acid and analyzed by ICP–MS. Error bars represent one standard deviation (n = 4).

Fig. 4

1/T₁ values of HEK 293T cells treated with 500 mM CG2 or Arg₈CG2 following incubation with vehicle control (black), copper (light gray, 100 μM, 48 h) or BCS (dark gray, 200 μM, 48 h). Relaxivity measurements were acquired at 37 °C in PBS (pH 7.4) at a proton Larmor frequency of 60 MHz. Error bars represent one standard deviation (n = 3) and statistical analyses were performed with a two-tailed Student’s t-test relative to the control. * P < 0.05, ** P < 0.01.

Fig. 5

1/T₁ values of MCH58 (black) and WG1005 (gray) cells treated with 500 μM CG2 (left) or Arg₈CG2 (right) following incubation with vehicle control, copper (100 μM, 48 h) or BCS (200 μM, 48 h). Relaxivity measurements were acquired at 37 °C in PBS (pH 7.4) at a proton Larmor frequency of 60 MHz. Error bars represent one standard deviation (n = 3) and statistical analysis were performed with a two-tailed Student’s t-test relative to the control. * P < 0.05, ** P < 0.01.

Fig. 6

T₁-weighted phantom images of MCH58 and WG1005 cells treated with CG2 or Arg₈CG2 (500 μM, 1 h). Images were acquired at 25 °C at 1.5 T (~64 MHz proton Larmor frequency).

Scheme 1

Copper-Gad contrast agent, CG2.

Scheme 2

Synthesis of Arg₈CG2.