Lanthanide complexes with acetophenone-based push-pull antennas as efficient MRI and two-photon microscopy imaging probes

Abstract

Lanthanide(iii) (Ln³⁺) complexes possess unique magnetic and optical properties that make them ideal candidates for the development of multimodal MRI and optical probes. However, the requirements for developing effective MRI and optical probes are difficult to meet within a single ligand. Here, we propose the use of a DOTA-type ligand equipped with a π-extended acetophenone moiety that (i) serves as a coordinating moiety to form stable Ln³⁺ complexes, (ii) acts as a 2P-excitable push-pull antenna to sensitize Eu³⁺ luminescence, and (iii) displays a carboxylate handle offering high water solubility and enabling conjugation to biomolecules for targeting purposes. We show that the Ln³⁺ complexes obtained are kinetically inert and thermodynamically stable. The Gd³⁺ complex exhibits positive in vivo characteristics with good contrast in all organs and rapid renal clearance, while the corresponding Eu³⁺ complex has excellent one-photon and two-photon (2P) absorption properties enabling high-quality in vivo 2P microscopy imaging of zebrafish embryos or in vitro imaging of living cells when conjugated to a cell-penetrating peptide.

Fig. 1. Structure of (A) complexes LnDO3A-AP^ArOR (Ln = Eu or Gd), EuDO3Apic^ArOR, EuDO3Apic^CCArOR, GdDO3A-AP, GdHPDO3A, GdDOTA, GdDO3A and Vasovist, all discussed in the text and (B) conjugate mTAT[EuDO3A-AP^ArOR].

Fig. 2. Synthetic pathway for (A) EuDO3A-AP^ArOR and GdDO3A-AP^ArOR and (B) mTAT[EuDO3A-AP^ArOR]. In (B), * denotes standard side chain protecting groups.

Fig. 3. (A) 1P spectroscopy: normalized absorption (black solid lines), excitation (black dashed lines; λ_em = 615 nm) and emission (red solid lines; λ_ex = 340 nm) spectra of EuDO3A-AP^ArOR in PBS, pH 7.4. (B and C) 2P spectroscopy: (B) quadratic power dependence of the Eu³⁺ emission (λ_ex = 700 nm) for EuDO3A-AP^ArOR in PBS pH 7.4; data were fitted using I = A × Pⁿ yielding n = 2.01; (C) 2P absorption spectrum (red) measured in PBS superimposed to the wavelength-doubled 1P absorption spectrum (black).

Fig. 4. (A) ¹H NMRD profiles of GdDO3A-AP^ArOR (0.88 mM) at 25 °C (blue), 37 °C (orange), and 50 °C (red). (B) Temperature dependence of the ¹⁷O reduced transverse relaxation rates (top) and chemical shifts (bottom) of GdDO3A-AP^ArOR at 9.4 T (9 mM). The dotted lines represent the simultaneous fit of the experimental data points.

Fig. 5. T ₁-Weighted phantom images in mouse serum with GdDO3A-AP^ArOR and GdDOTA (0.3 mM) at room temperature. Images were acquired at 7 T using a spin echo sequence with TE = 10 ms and TR = 400 ms. The intensities are respectively 3.89 × 10⁴, 3.97 × 10⁴ and 1.04 × 10⁴ for GdDO3A-AP^ArOR, GdDOTA, and serum.

Fig. 6. (A) T₁-weighted MR images recorded at 9.4 T of healthy mice pre-injection (left) and 2, 30 and 40 minutes post i.v. injection of GdDO3A-AP^ArOR at 100 µmol kg⁻¹, (B) % of MRI signal enhancement in the spleen, liver, kidneys and muscle, 2, 5, 10, 20, 30, 40 and 50 minutes post i.v. injection of GdDO3A-AP^ArOR at 100 µmol kg⁻¹ (n = 5, ±SD). (C) Gd content in the blood, spleen, liver, kidneys, and pancreas measured by ICP-OES 1 h after injection of GdDO3A-AP^ArOR (blue), GdDOTA (grey), or Vasovist (orange). Data are presented in % of injected dose by organ mass (n = 5, ±SD).

Fig. 7. Casper zebrafish embryo development expressed as percentages of chorionated, hatched, malformed and dead (A) without injection or after 1 nL of injection of (B) PBS or (C) EuDO3A-AP^ArOR (1 mM) at the single cell stage. The observation was carried out after 24, 48, 72, 96 and 120 hpi. The total number of embryos is 20, 15 and 20 for non-injected, PBS and EuDO3A-AP^ArOR respectively. Data are presented as mean ± SEM. (D) Representative images of the microscopic observation of embryos at 48 and 96 hpi using the microscope Zeiss Stemi 508.

Fig. 8. (A) 2P microscopy imaging of 72 hours post-fertilization of casper zebrafish embryos intravenously injected with 10 nL of EuDO3A-AP^ArOR (1 mM) and observed 3 h after injection at 720 nm (n = 5 embryos). Scale bar 500 µm. (B) Mean emission spectra in area outlined in blue and green in panel (A.) (C) Linear unmixing of autofluorescence and the Eu³⁺ signal.

Fig. 9. 2P microscopy imaging (λ_ex = 720 nm) of living HeLa cells incubated 1 h with mTAT[EuDO3A-AP^ArOR] (5 µM) and dFFLIPTAT (1.5 µM) in RPMI medium. (A) Left panel: differential interference contrast (DIC) image; middle panel: luminescence image recorded with 575–680 nm bp APD detection; right panel: merge. Scale bars correspond to 10 µm. (B) 2P-excited emission spectra (detected with a PMT array and averaged over the whole cell surface) of the cells outlined in red and blue and background (green). (C) Eu³⁺ luminescence decay in the cell outlined in red.