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.

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Introduction

Over the past number of decades, lanthanide complexes have been implemented with great success in various modalities in the field of biological imaging. Luminescent lanthanide complexes possess many advantageous properties for optical techniques, such as narrow emission bands at fixed wavelengths that are specific to each lanthanide, long luminescence lifetimes, and resistance to photobleaching.^1,2 Indeed, there are numerous examples of luminescent lanthanide complexes, such as those containing Eu³⁺, Tb³⁺ or Yb³⁺, being exploited for not only cellular imaging^3–8 but also for detection of various analytes.^3,9–12 Magnetic resonance imaging (MRI) is an imaging technique, endowed with unlimited tissue depth penetration and excellent spatiotemporal resolution. The exploitation of Gd³⁺-based contrast agents (CAs) in MRI has allowed for improved diagnostic accuracy¹³ and having the option to incorporate targeting moieties into the CA¹⁴ or render it “responsive”,^12,15,16 enabling the selective detection of various biologically relevant species – including but not limited to cations,^17–19 extracellular proteins,²⁰ and neurotransmitters.²¹

These two imaging modalities are not without their own drawbacks, however. While luminescence techniques display high sensitivity, they suffer from low macroscopic resolution and often require the integration of a cell-internalization vector, such as a peptide, to penetrate into living cells. Meanwhile, MRI has high macroscopic resolution but low sensitivity, requiring high concentrations of CAs to be detected. In recent years, the development of imaging probes active in both modalities has seen increased traction.^22–31 By incorporating complementary imaging modalities within a single molecular design, one could achieve non-invasive, real-time monitoring with high sensitivity and resolution, effectively overcoming the limitations of individual imaging modalities.^24,27,28 This would also have the benefit of complementary imaging at different scales, enabling monitoring of cellular processes and the anatomical context in real time.

Lanthanide complexes are particularly well-suited for these applications because of their different optical and magnetic properties across the series while still maintaining similar coordination properties and chemical reactivities. However, the development of pairs of lanthanide complexes relying on the same ligand, but displaying both MRI and luminescent activities is not without its own set of hurdles, either. MRI CAs require at least one inner-sphere coordinated water molecule to the Gd³⁺-center for efficient reduction of the T₁ relaxation time of water protons. This can be seen as incompatible with luminescent complexes which usually display a saturated coordination sphere, as directly coordinated water molecules quench lanthanide luminescence due to non-radiative deactivation.

Moreover, luminescent lanthanide complexes suffer from low molar absorption coefficients with values of about 1 to 5 M⁻¹ cm⁻¹, due to the Laporte rule that forbids the f–f transition. Nevertheless, this can be bypassed by the well-documented ‘antenna effect’, whereby a chromophore is implemented close to the lanthanide.^2,12,32 This chromophore should efficiently absorb light, typically in the ultraviolet (UV) range, followed by subsequent energy transfer to the lanthanide, thus allowing for the sensitization of the lanthanide and resulting luminescence emission. It is important to note that the use of UV light to excite the antenna is problematic, as UV light has not only been proven to be cytotoxic, but can also be absorbed and scattered by biological tissues. An alternative to this excitation pathway is the use of a two-photon (2P) absorption process that allows for the simultaneous absorption of two photons, with half the energy required for a normal excitation with one photon.^33–36 This method allows for a bathochromic shift of the excitation wavelength, moving from the UV to the red or near infrared (NIR) region, which is known to be less toxic and less scattered by biological tissues. Unlike MRI, one of the main drawbacks of luminescence is its low tissue penetration. 2P excitation in the NIR region allows deeper tissue penetration, thus compensating for the limitation of fluorescence imaging.^37,38

We have recently developed 2P excitable Ln³⁺ probes that were used successfully for 2P microscopy of live cells.^39–43 These probes are based on a DO3Apic ligand that saturates the Ln³⁺ coordination sphere, precluding their use as MRI CAs. With the aim to design an octadentate Ln³⁺ chelator that leaves a single water molecule in the coordination sphere of the Ln³⁺ and features a 2P absorbing antenna, we identified DO3A-AP in the literature. This ligand was first reported to bind Eu³⁺ and sensitize its luminescence through the acetophenone antenna.⁴⁴ EuDO3A-AP is mono-hydrated and shows a Eu³⁺ luminescence quantum yield of 0.058 upon excitation in the acetophenone absorption band at 265 nm. The absorption could be red-shifted by introducing electron-rich methoxy⁴⁴ or triazole⁴⁵ groups in the para-position of the acetophenone or by substituting the phenyl-ketone by other aryl-ketones (aryl = naphthyl, carbazolyl or phenantrenyl).⁴⁶ The relaxometric properties of GdDO3A-AP and related derivatives with hydroxy substituents were recently described.^47,48 However, the in vivo MRI (with Gd³⁺ as Ln³⁺) or in vitro/in vivo microscopy imaging (with Eu³⁺) capacities of the LnDO3A-AP system were never explored.

Based on this, we wanted to push the advantage and propose for the first time a monohydrated Ln³⁺ complex for both high resolution 2P microscopy, with Eu³⁺ as Ln³⁺, and MRI, with Gd³⁺. We present here DO3A-AP^ArOR (Fig. 1), a ligand derived from DO3A-AP bearing an antenna with strong push–pull capabilities providing 2P absorption properties and a reactive handle for the facile incorporation of a cell-penetrating peptide. The coordination sphere remains unsaturated, allowing for one water molecule in the inner-sphere, providing the opportunity for its use as an MRI contrast agent. The photophysical properties of the Eu³⁺ complex were investigated, alongside its 2P excitation properties, and compared to those of the previously described non-hydrated picolinate analogues EuDO3Apic^ArOR and EuDO3Apic^CCArOR.^39,43 Despite its coordinated water molecule, EuDO3A-AP^ArOR shows good luminescence properties. The relaxometric properties of the Gd³⁺ complex were characterized by variable temperature ¹⁷O NMR experiments and nuclear magnetic relaxation dispersion. The complex also displays very high kinetic inertness. These positive features allow for the in vivo use of the Gd³⁺ complex as an efficient MRI contrast agent and the Eu³⁺ complex as a luminescent probe for 2P microscopy in zebrafish. A cell-penetrating peptide, mTAT, was conjugated to the antenna which allowed for internalization of the complex to the cytosol of live HeLa cells, visualized by 2P microscopy. Therefore, this versatile design based on a Ln³⁺ complex allows in vivo MRI in mice and 2P microscopy on zebrafish and high quality cell imaging by 2P microscopy.

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].

Results and discussion

Synthesis of the ligand and its lanthanide complexes

The synthetic pathway for the preparation of the acetophenone-based Ln³⁺ complexes is depicted in Fig. 2A. The synthesis starts with the alkylation of DO3A(tBu)₃ 1 (ref. 49) with 2-bromo-1-(4-iodophenyl)ethan-1-one 2 in MeCN to give compound 3 in 94% yield. A Miyaura–Suzuki coupling between 3 and boronic ester 4 (ref. 43) in DMF with polymer-bound Pd(PPh₃)₄ as a catalyst affords the tBu-protected ligand 5 in 56% yield. After acidolysis of the tBu esters in a TFA/DCM mixture, metalation with Eu³⁺ or Gd³⁺ salt in water (pH 7) followed by HPLC purification and freeze-drying gives complexes LnDO3A-AP^ArOR in 80–90% yield. Note that these compounds are sensitive to basic conditions, which can cause N-dealkylation of the acetophenone arm. The detailed synthetic procedures and NMR, HPLC and mass spectrometry characterization of these compounds are given in the SI.

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.

Photophysical properties

The photophysical properties of the EuDO3A-AP^ArOR complex were investigated in phosphate-buffered saline (PBS, pH 7.4). The absorption spectrum of EuDO3A-AP^ArOR shows a broad structureless band in the UV region with a maximum at 342 nm (λ_max) extending up to 403 nm (λ_cut-off), which is assigned to an intra-ligand charge transfer transition (ILCT) within the antenna from the anisole donor to the coordinated carbonyl acceptor moieties (Fig. 3). This absorption is red-shifted by ca. 35 nm and 10 nm (Fig. S8) compared to the picolinate analogues EuDO3Apic^ArOR and EuDO3Apic^CCArOR (Fig. 1) with alkoxy-phenyl-picolinate and alkoxy-phenyl-ethynyl-picolinate antennas, respectively.^39,43 The molar absorption coefficient of EuDO3A-AP^ArOR was determined to be 20 000 M⁻¹ cm⁻¹ at λ_max (Fig. S9) similar to EuDO3Apic^ArOR (21 000 M⁻¹ cm⁻¹)⁴³ and EuDO3Apic^CCArOR (21 000 M⁻¹ cm⁻¹).³⁹ Upon excitation into the ILCT band at 340 nm, the Eu³⁺ emission is observed with ⁵D₀ → ⁷F_J transitions (J = 0, 1, 2, 3 and 4) at 580, 595, 615, 650 and 700 nm, respectively. The excitation spectrum (λ_em = 615 nm) matches well with the absorption spectrum, confirming that the alkoxy-phenyl-acetophenone antenna sensitizes Eu³⁺ luminescence. The Eu³⁺ luminescence decay could be perfectly fitted with a mono-exponential giving a lifetime value of 0.53 (2) ms (Fig. S10), which is about half that of the picolinate analogues EuDO3Apic^ArOR and EuDO3Apic^CCArOR (1.1 ms) with a saturated Eu³⁺ coordination sphere (hydration number q = 0).^39,43 The lifetime remains unchanged upon degassing the solution. In PBS prepared with D₂O, the Eu³⁺ decay lifetime was 1.49 ms. According to Parker's equation, a hydration number q of 1.2 (2) was calculated from the lifetimes in H₂O and D₂O,⁵⁰ indicating that a single water molecule stands in the coordination sphere of Eu³⁺. This is in agreement with the literature on Ln³⁺ complexes of DO3A-AP ligands.^44–48 The Eu³⁺ emission quantum yield, Φ_Eu, is 0.075 (Fig. S11), again almost half that of the picolinate analogues (0.14 and 0.17 for EuDO3Apic^ArOR and EuDO3Apic^CCArOR, respectively).^39,43 The energy of the alkoxy-phenyl-acetophenone antenna triplet state, T₁, is 21 100 cm⁻¹ (determined from the onset of the phosphorescence emission of GdDO3A-AP^ArOR at 77 K), which is 2000 cm⁻¹ below that of the picolinate analogue (Fig. S12).⁴³ However, the antenna T₁ state remains high enough to prevent efficient back energy transfer from the Eu^{3+ 5}D₀ excited state. In the end, EuDO3A-AP^ArOR is half as bright as its picolinate analogue because of the presence of one Eu³⁺-bound water molecule.

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).

The 2P absorption properties of EuDO3A-AP^ArOR were determined by the two-photon excited fluorescence method. First, the quadratic dependence of the emitted Eu³⁺ intensity on laser power was checked under excitation at 700 nm with a Ti:sapphire laser (Fig. 3B). Then, the 2P absorption spectrum was measured. It matches nicely the wavelength-doubled 1P absorption spectrum (Fig. 3C). At 720 nm, the wavelength used in 2P microscopy experiments (vide infra), the 2P absorption cross-section, σ_2P, is 36 GM (1 GM = 10⁻⁵⁰ cm⁴ s photon⁻¹), similar to that of the alkoxy-phenyl-ethynyl-picolinate antenna (35 GM)⁵¹ and ca. 10 times higher than that measured for an Eu³⁺ complex with a methoxy-phenyl-picolinamide antenna.⁴² At 720 nm, the 2P brightness, B_2P = σ_2P × Φ_Eu, of EuDO3A-AP^ArOR is 2.7 GM.

Relaxometric characterization of GdDO3A-AP^ArOR

Since EuDO3A-AP^ArOR has a hydration number q of 1, its Gd³⁺ analogue appears to be a good candidate for MRI imaging. In order to characterize the efficacy of GdDO3A-AP^ArOR and relate this to the microscopic parameters that govern the efficacy (relaxivity) of the complex, nuclear magnetic relaxation dispersion (NMRD) profiles, in combination with variable temperature ¹⁷O NMR experiments, were recorded. The NMRD profiles were recorded within the range of 0.01–600 MHz, at three different temperatures: 25, 37, and 50 °C (Fig. 4A). As temperature increased, the r₁ values decreased, indicating that the rotational correlation time is the limiting factor in the relaxivity, which is expected with a small molecular complex. The relaxivity at 20 MHz, 25 °C is 5.4 mM⁻¹ s⁻¹, consistent with a monohydrated complex of this size and aligns well with the values found under the same conditions for GdHPDO3A (4.6 mM⁻¹ s⁻¹)⁵² and GdDO3A-AP (5.1 mM⁻¹ s⁻¹).⁴⁷

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.

The reduced transverse relaxation rates (1/T_2r) were measured as a function of temperature to determine the water exchange rate, k_ex, while the reduced chemical shifts give access to the number of coordinated water molecules on the Gd³⁺ complex. The ¹⁷O-reduced transverse relaxation rates increase (up to ca. 333 K), followed by a plateau (Fig. 4B). The shape of the curve indicates that there are at least two isomers present in solution, most likely the SAP and TSAP coordination isomers which display greatly different water exchange rates, as seen with GdHPDO3A,⁵² GdDOTA bisamide derivatives^53,54 or GdDO3A-AP.⁴⁷ Thusly, a ¹H NMR study was performed (700 MHz at 288, 298, and 318 K) to determine the ratio between the SAP and TSAP isomers of EuDO3A-AP^ArOR in solution (Fig. S14). The SAP isomer is found to be the main isomer as already reported for the majority of DOTA-based complexes.⁵⁵ The ratio of SAP : TSAP is found to be 80 : 20, which is consistent with complexes reported in the literature with a similar coordination sphere.^46,47 Importantly, this ratio is constant over the range of temperature studied. The reduced chemical shifts measured are consistent with a monohydrated complex in solution, in accordance with the luminescence measurements on the Eu³⁺ complex.

The NMRD profiles, transverse ¹⁷O relaxation rates, and ¹⁷O chemical shifts were fitted simultaneously using Solomon-Bloembergen and Morgan (SBM) theory, while considering the relative populations of the two isomers in solution, to yield the microscopic parameters that govern the proton relaxivity of the complex (Tables 1 and S1). The NMRD profiles were fitted in the range 4–600 MHz, where the effect of the electron spin relaxation, which is not well described by the SBM theory alone, is negligible, and the SBM approach gives reliable information on dynamic processes like the water exchange rate and rotational correlation time for small complexes.^56,57

Table 1 The best-fit parameters obtained from the simultaneous fitting of the NMRD profiles at 298 K, 310 K, and 323 K, and the transverse ¹⁷O relaxation rates and chemical shifts as a function of temperature at 9.4 T

	Isomer	GdDO3A-AP^ArOR	GdDO3A-AP^47	GdHPDO3A^52
r1 (mM^−1 s^−1) 20 MHz, 25 °C		5.4	5.1	4.6
kex^298 (10^6 s^−1)	SAP	0.49 (3)	0.83	1.56
ΔH^≠ (kJ mol^−1)	SAP	55 (3)	50	53
ΔS^≠ (kJ mol^−1 K^−1)	SAP	+49	+36	+52
kex^298 (10^6 s^−1)	TSAP	18 (3)	40	112
ΔH^≠ (kJ mol^−1)	TSAP	42 (8)	27	15
τR^298 (ps)		134 (3)	100	65

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Under the reasonable assumption that the rotational correlation time (τ_R) is the same for both SAP and TSAP species, a value of 134 ps is found. This value is higher than that of GdHPDO3A (65 ps) and GdDO3A-AP (100 ps), which is consistent with the larger size of GdDO3A-AP^ArOR. The fitting of the data yields k_ex = 0.49 × 10⁶ s⁻¹ for the SAP isomer and k_ex = 18 × 10⁶ s⁻¹ for the TSAP isomer. This is a well-known phenomenon, whereby the increased steric hindrance around the Gd³⁺-center of the TSAP isomer results in a faster water exchange rate. It was previously observed that the introduction of a ketone pendant arm resulted in slow water exchange rates.^47,58 In this case, the exchange rates of the SAP and TSAP isomers are even slower than those of GdDO3A-AP. This could be explained by the electronic effects of the benzene on the acetophenone, which decreases the electronic density on the ketone; therefore the steric crowding around Gd³⁺. In such complexes with positive activation entropy (dissociatively activated mechanism), this results in a lower water exchange rate.⁵⁹

Due to the presence of the hydrophobic biphenyl moiety, the complex could be prone to aggregation processes. Paramagnetic Relaxation Enhancements (PRE) measurements were performed at 60 MHz and 25 °C as a function of concentration (Fig. S15). A linear trend is observed, which demonstrates no aggregation of the complex in the concentration range studied (ca. 80 µM–8 mM).

The incorporation of a lipophilic group into a Gd³⁺-based contrast agent is a well-established method to promote non-covalent interactions between the CA and human serum albumin (HSA). This interaction has several advantages, such as increased relaxivity of the CA, mainly due to the increased rotational correlation time of the chelate, and a prolonged vascular retention time. Considering that biphenyl moieties have been leveraged multiple times to this effect, we considered that the biphenyl antenna present in DO3A-AP^ArOR could possibly interact with HSA in this manner. Thus, the relaxivity of GdDO3A-AP^ArOR in the presence of 0.6 mM HSA was measured at 60 MHz, 25 °C. An increase of ca. 110% in relaxivity was observed in the presence of HSA (r₁ = 11.4 mM⁻¹ s⁻¹ vs. r₁ = 5.38 mM⁻¹ s⁻¹ in HEPES buffer).

To further evaluate the potential of this complex as a contrast agent, T₁-weighted phantom images were recorded at 7 T, where the relaxivity of GdDO3A-AP^ArOR was measured in mouse blood serum and compared to that of GdDOTA, a Gd³⁺-based CA used clinically (Fig. 5). While GdDO3A-AP^ArOR did not retain its enhanced relaxivity at higher field strengths, it did display a similar signal intensity (3.89 × 10⁴) to the clinically used GdDOTA (3.97 × 10⁴) at 7 T, 25 °C (0.3 mM in mouse serum).

Fig. 5 T₁-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.

Stability

Given the in vitro potential of the system both in terms of luminescence and relaxivity, we wanted to assess its in vivo applicability. However, it is of prime importance to check the thermodynamic stability and kinetic inertness of the system prior to in vivo use. Macrocyclic lanthanide complexes based on DOTA or DOTA-monoamide systems are known to display high thermodynamic stability and kinetic inertness.⁶⁰ In this case, one ketone function is coordinating the Ln³⁺. Therefore, we first investigated the thermodynamic stability and kinetic inertness of EuDO3-AP^ArOR by competitions with DTPA at pH 7.4. EuDO3A-AP^ArOR was mixed with DTPA (1, 10 and 100 eq.) at pH 7.4 in PBS and the Eu³⁺ emission intensity was monitored after 24 h. At 1 : 1 and 1 : 10 EuDO3A-AP^ArOR/DTPA ratios, the Eu³⁺ emission intensity remained unchanged (Fig S16). A slight decrease in Eu³⁺ intensity was observed for a 1 : 100 ratio. This is indicative of a high thermodynamic stability or kinetic inertness. Under more challenging conditions to assess the kinetic inertness and compare to other macrocyclic complexes, EuDO3-AP^ArOR was dissolved in HCl 1 M. The emission spectrum is the same as that at pH 7.4 (Fig. S17A), indicating that the coordination sphere of the emissive Eu³⁺ species is not altered by the low pH. Under these conditions, the complex fully dissociates (Fig. S17B). Given the very high proton concentration, the dissociation follows a pseudo-first order kinetics, and the dissociation rate is proportional to the total concentration of the complex [LnL]_t, with k_obs being the pseudo-first order rate constant:

The dissociation half-life and k_obs values determined in 1 M HCl are presented in Table 2. The k_obs is one order of magnitude higher than that of GdDOTA,⁶¹ but two to three orders of magnitude lower than that of the other macrocyclic compounds (GdDO3A⁶² or EuDO3Apic⁶³). Importantly, it is one order of magnitude lower than that of commercially available GdHPDO3A.⁶⁴ The half-life in 1 M HCl is 5.3 h, which demonstrates the strong inertness of EuDO3-AP^ArOR.

Table 2 Rate constants characterizing the dissociation of complexes determined in 1 M HCl at 298 K and half-life under the same conditions

	kobs (s^−1)	t1/2 (min)
EuDO3A-AP^ArOR	(3.6 ± 0.2) × 10^−5	320 ± 20
GdDOTA^61	1.8 × 10^−6	6418
GdDO3A^62	2.3 × 10^−2	0.5
EuDO3Apic^63	2.0 × 10^−3	5.7
GdHPDO3A^64	2.6 × 10^−4	44

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In vivo MRI biodistribution in mice

Encouraged by the in vitro results and the very high inertness of the complex, an in vivo MRI study was then carried out, where five healthy mice were injected with GdDO3A-AP^ArOR (100 µmol kg⁻¹). After intravenous administration of the contrast agent, an immediate increase in signal intensity was observed, with the signal enhancement peaking two minutes after injection for all organs (Fig. 6). The kidneys experienced the greatest signal enhancement over other organs. This indicates that the CA is cleared via the kidneys, with a negligible portion via the liver, which is expected for a small molecular complex. The clearance is fast as the signal enhancement in the main organs (liver, spleen and muscle) is negligible 50 min post-injection. Ex vivo biodistribution was also studied 1 h post injection and the Gd³⁺ content of various organs was measured ex vivo by Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) (Fig. 6). Less than 3% of the injected dose per organ gram is found, showing a good elimination of the contrast agent after one hour. Interestingly, the Gd content one hour post injection is intermediate to that found for GdDOTA and Vasovist,⁶⁵ a blood-pool agent. This is consistent with the in vitro results showing some HSA binding and a certainly longer circulation time than GdDOTA, but shorter than Vasovist. The excellent stability and inertness of GdDO3A-AP^ArOR and successful MRI experiments prompted us to evaluate the potential of the Eu³⁺ analogue EuDO3A-AP^ArOR for in vivo 2P microscopy imaging in zebrafish embryos.

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).

In vivo 2P microscopy of zebrafish embryos

The use of zebrafish (Danio rerio) embryos is increasingly recognized as an alternative to testing on small mammals.^66,67 Although imaging of zebrafish embryos with fluorescent probes is classical,⁶⁸ to our knowledge, examples of 2P zebrafish imaging with Ln³⁺ probes are scarce.^7,69 First, the toxicity of EuDO3A-AP^ArOR in zebrafish embryos was studied at the single cell stage (see the SI for details). The development of embryos was observed at different time points (24, 48, 72, 96 and 120 h) post-injection. The viability was based on the morphology and the presence of heartbeat. Malformation including pericardial oedema, yolk sac oedema, spinal curvature and tail malformation was recorded. Dead embryos were removed immediately. The results shown in Fig. 7 demonstrate the mild toxicity of EuDO3A-AP^ArOR in vivo (Fig. 7c). At 72 hpi (hours post-injection), an increase in the death rate of the EuDO3A-AP^ArOR injected group (25%) compared to the non-injected group (5%) was observed. Furthermore, an increase in the hatching rate in the PBS and EuDO3A-AP^ArOR injected groups (∼70%) compared to the non-injected group (25%) was recorded. EuDO3A-AP^ArOR did not induce higher morphological abnormalities than the non-injected or PBS injected group. The toxicity profile of EuDO3A-AP^ArOR remains constant from 72 hpi until 120 hpi.

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.

Then, 2P microscopy imaging of zebrafish embryos was performed under 720 nm excitation with spectral detection for 3 h after intravenous injection of EuDO3A-AP^ArOR (see the SI for details). As shown in Fig. 8A, the luminescence signal was collected with a 570–640 bp filter and a long integration time of 131 µs per pixel. The emission was detected in the heart, intersegmental vessels, caudal vein, caudal artery, primary head sinus and the inner optic circle. In contrast, in the control (no Eu³⁺ probe injected), the luminescence was detected mainly in the yolk, which usually shows high autofluorescence. In injected embryos, spectral detection (Fig. 8B) allowed for the discrimination of the broad autofluorescence signal with a maximum at ca. 460 nm and the Eu³⁺ emission with characteristic peaks at 580 nm and 620 nm. As expected, the latter was not detected in the control. Linear unmixing of the autofluorescence and Eu³⁺ signals is shown in Fig. 8C. EuDO3A-AP^ArOR is clearly distributed in the cardiovascular circulatory system. Finally, the viability of embryos injected with EuDO3A-AP^ArOR was 80% after 3 days post-injection, confirming its low toxicity. Therefore, EuDO3A-AP^ArOR is suitable to provide high-quality 2P images in zebrafish.

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.

Preparation and characterization of the TAT conjugate

The in vivo applicability of the complex is well established in both MRI and luminescence to probe the extracellular environment. However, for bioimaging applications, it can be interesting to specifically vectorize the probe, for example, towards cancer cells or to make it cell-permeable in order to probe the intracellular environment and obtain complementary information. This is generally achieved by coupling the probe to a peptide or a protein that provides recognition properties. Here, we choose to demonstrate the applicability of EuDO3A-AP^ArOR for 2P microscopy at the cell level by conjugating it to the well-known TAT cell penetrating peptide.^41,42,70 The preparation of TAT conjugates with LnDO3Apic complexes, which were described previously, relies on the coupling of a protected macrocyclic ligand to the peptide on resin, followed by (i) acidic cleavage from the resin and deprotection (side chains and ligand), (ii) HPLC purification and (iii) metalation with Ln³⁺. Here we opted for a more straightforward strategy, directly coupling the Ln³⁺ complex to the peptide on resin (Fig. 2B). The peptide was elongated on Rink Amide resin using solid phase peptide synthesis following classical procedures for the Fmoc/tBu strategy. The N-terminal lysine was orthogonally protected with an alloc group. After selective removal of this group on resin using Pd⁰, EuDO3A-AP^ArOR was coupled to the peptide via its carboxylate handle using HATU/DIEA activation for 2 h. After resin cleavage and protecting group removal using TFA/H₂O/TIS, the conjugate was purified by HPLC and freeze-dried. The Eu³⁺ complex resisted the TFA treatment and the acidic HPLC conditions (H₂O/MeCN mixture with 0.1% TFA, pH ca. 2) and no Eu³⁺ release was observed, further validating the high inertness of the complex. Proper conjugation through the carboxylate handle on the electron donating group of the antenna was confirmed by mass spectrometry and Eu³⁺ luminescence properties.

The spectroscopic properties of mTAT[EuDO3A-AP^ArOR] are very similar to those of the parent Eu³⁺ complex (Fig. S13), with an identical absorption band, the same Eu³⁺ emission spectrum, which attests that the conjugation has not altered the Eu³⁺ coordination sphere, and identical Eu³⁺ luminescence lifetime (0.50 (2) ms) and quantum yield (Φ_Eu = 0.077) within the error margin. All this indicates that conjugation to the TAT peptide has no influence on the emission properties of EuDO3A-AP^ArOR.

In vitro 2P microscopy on living HeLa cells

In vitro imaging was attempted with live HeLa cells incubated with a mixture of mTAT[EuDO3A-AP^ArOR] and dFFLIPTAT, a dimeric cell penetrating peptide (sequence: (CFFLIPRKKRRQRRRG)₂, dimerized via a disulfide bond) that helps TAT monomers such as mTAT[LnL] probes to enter live cells.^41,42,71 Before conducting 2P microscopy on live HeLa cells, the cytotoxicity of the conjugate in co-incubation with dFFLIPTAT (1.5 µM) was examined using the MTT assay (Fig. S18). After 48 h of cell proliferation, no cytotoxicity was observed when the conjugate was incubated at 5 µM and 10 µM.

For microscopy experiments, HeLa cells were incubated with mTAT[EuDO3A-AP^ArOR] (5 µM) and dFFLIPTAT (1.5 µM) for 1 h before washing and imaging on a 2P microscope under 720 nm excitation using an APD (avalanche photodiode) for sensitive detection and a 580–680 nm bandpass filter to collect Eu³⁺ emission Fig. 9A. The DIC (differential interference contrast) image shows HeLa cells with a phenotype of living cells in agreement with the MTT assay. The luminescence channel shows stained cells with diffuse red emission within the whole cell, typical of successful cytosolic delivery of the probe.^40,42 The cell-to-cell intensity heterogeneity is a common feature of probes based on cell penetrating peptides.^40,42,72,73 In order to confirm that the luminescence channel collects Eu³⁺ emission, a spectral image was recorded using the PMT array of the 2P microscope. Fig. 9B shows the mean emission spectra of cells, recorded over the area outlined in red and blue in the DIC image. For the ⁵D₀ → ⁷F_J, J = 1 and 2, emission bands of Eu³⁺ emission are unambiguously observed between 575 and 630 nm. The Eu³⁺ luminescence lifetime was measured in cells using the TSLIM method (Fig. 9C).⁷⁴ A value of 0.57 ± 0.03 ms (average over 15 measurements in three distinct cells) was found, similar to the one recorded in solution. This was surprising because with other Eu³⁺ or Tb³⁺ probes featuring chelators that saturate the Ln³⁺ coordination sphere, we have always measured lifetimes ca. 30% shorter in cells than in the cuvette.^40,75 This shorter lifetime was proposed to be the consequence of additional de-excitation pathways in the cell via protein co-factors. In the present case, the higher lifetime suggests a change in the average hydration number of Eu³⁺, i.e. a partial replacement of the Eu³⁺-bound H₂O by coordinating amino acid side chains of a protein or small molecule. Note that under similar incubation conditions (1 h, 5 µM probe), the non-conjugated complex EuDO3A-AP^ArOR is not detected within cells. Addition of DMSO (up to 5%) in the incubation medium to permeabilize membranes did not improve uptake. This demonstrates the value of conjugating the probe to a cell penetrating peptide. To sum up, despite its Eu³⁺-bound water molecule, EuDO3A-AP^ArOR conjugated to the TAT cell penetrating peptide allows 2P microscopy of live cells and gives high-quality images of live cells.

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.

Conclusions

In this article, we have demonstrated that adding a π-conjugated extension to the acetophenone moiety of ligand DO3A-AP provides a luminescent Eu³⁺ complex, EuDO3A-AP^ArOR, with excellent 2P absorption properties compared to its picolinate analogue. Despite an Eu³⁺-bound water molecule, which quenches Eu³⁺ emission and lowers luminescence properties, this complex has sufficiently good luminescence properties to make it suitable for in vivo 2P imaging of zebrafish and live cell 2P imaging. Compared to the picolinate analogue, the water molecule bound to Ln³⁺ proves to be an advantage, allowing MRI imaging in vivo with the Gd analogue, GdDO3A-AP^ArOR. This complex is therefore able to probe the extracellular environment in vivo both in MRI, with Gd³⁺, and 2P microscopy imaging, with Eu³⁺. Several DO3A- or DOTA-based ligand systems were already described to elaborate by simple Ln³⁺ exchange on both luminescent Eu³⁺ or MRI-active Gd³⁺ probes, with predictable MRI properties due to the DO3A/DOTA scaffold.^26–29 However, they were used in luminescence microscopy imaging with UV excitation. The system described here integrates a push–pull antenna that permits 2P excitation in the NIR region, which is less damaging and allows better tissue penetration. The strong 2P absorption properties of this system provided for instance excellent quality microscopy images of zebrafish embryos. Finally, another advantage of this system is its pending carboxylate, which allows straightforward conjugation of LnDO3A-AP^ArOR complexes to biomolecules such as peptides. This has been exemplified here with a TAT conjugate, a cell-penetrating peptide, allowing high-quality living cell microscopy images to be obtained following 2P absorption despite the presence of the water molecule on Ln³⁺. Therefore, LnDO3A-AP^ArOR complexes have great potential and versatility for the safe development of efficient agents for MRI and 2P microscopy imaging.

Ethical statement

All animal experiments were carried out in accordance with the guidelines for animal experiments and under permission number 54201, from the French “Ministère de l’Enseignement Supérieur, de la Recherche et de l’Innovation”.

Author contributions

Conceptualization: O. S., C. S. B., O. M., and M. G.-B.; investigation: B. C., L. M., L. M. A. A., D. A., G. M., S. M., S. E., V. M.-F., A. G., and O. S.; validation: O. S., C. S. B., M. G.-B., O. M., A. B., S. M. A. G., V. M.-F., and D. B.; writing (original draft): B. C., L. M., L. M. A. A., O. S., C. S. B., and M. G.-B.; writing (review & editing): all authors. visualization: O. S., C. S. B., B. C., L. M., and L. M. A. A.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information: procedures for the synthesis of new compounds, 1P and 2P spectroscopy (absorption and luminescence), relaxometry, stability and inertness assays by luminescence, protocols for MRI experiments, toxicity assays and 2P microscopy of zebrafish embryos, cell culture, toxicity assays and 2P microscopy. See DOI: https://doi.org/10.1039/d5sc06902e.

Acknowledgements

Agnès Pallier is acknowledged for performing ICP experiments. The authors thank the MO2VING platform for spectroscopic and MRI experiments. For zebrafish embryo imaging, the authors acknowledge the imaging facility MRI, a member of the France-BioImaging national infrastructure, supported by the French National Research Agency (ANR-10-INBS-04, “Investments for the future”) and also Nicolas Cubedo and Mireille Rossel from the aquatic model facility ZEFIX from MMDN/LPHI/CRBM/IGF. Cell 2P microscopy imaging experiments were done on the Microcell core facility of the Institute for Advanced Biosciences (UGA – Inserm U1209 CNRS 5309). This facility belongs to the IBISA-ISdV platform, a member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04). The authors acknowledge the Agence Nationale de la Recherche [LANTEN project (ANR-21-CE29-0018) and the Zinc-Espionage project (ANR-22-CE44-0041)], the Labex ARCANE and CBH-EUR-GS (ANR-17-EURE-0003) and the CEA FOCUS Biomarqueurs for financial support.

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