Investigation of Ln3+ complexation by a DOTA derivative substituted by an imidazothiadiazole: synthesis, solution structure, luminescence and relaxation properties.

We investigated the coordination properties of original macrocyclic Ln3+ complexes comprising an imidazothiadiazole heterocycle. The thermodynamic stability of the Gd3+ complex was determined by a combination of potentiometric and photophysical measurements. The kinetic inertness was assessed in highly acidic media. The solution structure of the Ln3+ complex was unambiguously determined by a set of photophysical measurements and 1H, 13C, 89Y NMR data in combination with DFT calculations, which proved coordination of the heterocycle to Ln3+. The ability of the imidazothiadiazole moiety to sensitize Tb3+ luminescence was investigated. Finally, the relaxation properties were investigated by recording 1H nuclear magnetic relaxation dispersion (NMRD) profiles and 17O measurements. The water exchange rate is similar to that of GdDOTA as the less negative charge of the ligand is compensated for by the presence of a bulky heterocycle. Relaxivity is constant over a large range of pH values, demonstrating the favorable properties of the complex for imaging purposes.

Lanthanide-Based Probes for Imaging Detection of Enzyme Activities by NIR Luminescence, T1- and ParaCEST MRI.

Applying a single molecular probe to monitor enzymatic activities in multiple, complementary imaging modalities is highly desirable to ascertain detection and to avoid the complexity associated with the use of agents of different chemical entities. We demonstrate here the versatility of lanthanide (Ln3+) complexes with respect to their optical and magnetic properties and their potential for enzymatic detection in NIR luminescence, CEST and T1 MR imaging, controlled by the nature of the Ln3+ ion, while using a unique chelator. Based on X-ray structural, photophysical, and solution NMR investigations of a family of Ln3+ DO3A-pyridine model complexes, we could rationalize the luminescence (Eu3+, Yb3+), CEST (Yb3+) and relaxation (Gd3+) properties and their variations between carbamate and amine derivatives. This allowed the design of L n L G a l 5 ${{{\bf L n L}}_{{\bf G a l}}^{5}}$ probes which undergo enzyme-mediated changes detectable in NIR luminescence, CEST and T1-weighted MRI, respectively governed by variations in their absorption energy, in their exchanging proton pool and in their size, thus relaxation efficacy. We demonstrate that these properties can be exploited for the visualization of ß-galactosidase activity in phantom samples by different imaging modalities: NIR optical imaging, CEST and T1-weighted MRI.

Zinc-sensitive MRI contrast agents: importance of local probe accumulation in zinc-rich tissues.

We present the in vitro characterisation of a Gd3+-based contrast agent that responds to Zn2+ upon interaction with Human Serum Albumin. We show that the contradictory in vivo behaviour is related to Gd3+-accumulation in Zn-rich tissues. This highlights the importance of the biodistribution of such contrast agents.

Gd3+ Complexes for MRI Detection of Zn2+ in the Presence of Human Serum Albumin: Structure-Activity Relationships.

Zn2+-responsive magnetic resonance imaging (MRI) contrast agents are typically composed of a Gd chelate conjugated to a Zn2+-binding moiety via a linker. They allow for Zn2+ detection in the presence of human serum albumin (HSA). In order to decipher the key parameters that drive their Zn2+-dependent MRI response, we designed a pyridine-based ligand, PyAmC2mDPA, and compared the properties of GdPyAmC2mDPA to those of analogue complexes with varying Gd core, Zn-binding moiety, or linker sizes. The stability constants determined by pH potentiometry showed the good selectivity of PyAmC2mDPA for Gd3+ (log KGd = 16.27) versus Zn2+ (log KZn = 13.58), proving that our modified Zn2+-binding DPA moiety prevents the formation of previously observed dimeric species. Paramagnetic relaxation enhancement measurements indicated at least three sites that are available for GdPyAmC2mDPA binding on HSA, as well as a 2-fold affinity increase when Zn2+ is present (KD = 170 µM versus KDZn = 60 µM). Fluorescence competition experiments provided evidence of the higher affinity for site II vs site I, as well as the importance of both the Zn-binding part and the Gd core in generating enhanced HSA affinity in the presence of Zn2+. Finally, an analysis of nuclear magnetic relaxation dispersion (NMRD) data suggested a significantly increased rigidity for the Zn2+-bound system, which is responsible for the Zn2+-dependent relaxivity response.

A phosphorylated zinc finger peptide bearing a gadolinium complex for zinc detection by MRI.

Two zinc finger peptides, namely ZFQDLn and ZFQELn (Ln = Tb or Gd), with an appended Ln3+ chelate and a phosphoserine able to coordinate the Ln3+ ion are presented. The two peptides differ by the amino acid anchorage of the chelate, either aspartate (D) or glutamate (E). Both peptides are able to bind Zn2+ and adopt the ßßα fold. Interestingly, ZFQETb shows a decrease in sensitized Tb3+ luminescence upon Zn2+ binding whereas ZFQDTb does not. The luminescence change upon Zn2+ binding is attributed to a change in hydration number (q) of the Tb3+ ion due to the decoordination of the phosphoserine from the Ln3+ ion upon Zn2+ binding and peptide folding. This process is highly sensitive to the length of the linker between the Ln chelate and the peptidic backbone. The magnetic properties of the gadolinium analogue ZFQEGd were studied. An impressive relaxivity increase of 140% is observed at 60 MHz and 25 °C upon Zn2+ binding. These changes can be attributed to a combined increase effect of the hydration number of Gd3+ and of the rigidity of the system upon Zn2+ binding. Phantom MR images at 9.4 T show a clear signal enhancement in the presence of Zn2+. These zinc finger peptides offer a unique platform to design such Zn-responsive probes.

Insights into the Responding Modes of Highly Potent Gadolinium-Based Magnetic Resonance Imaging Probes Sensitive to Zinc Ions.

Zn ions (Zn2+) play an important biological role in many diseases; hence, an imaging method for monitoring the Zn2+ distribution in tissues could provide important clinical insights. Recently, we reported a potent Zn-sensitive probe based on the Gd-DO3A (DO3A = 1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylic acid), modified tyrosine. and di(2-picolyl)amine chelator for this metal cation, which generates an outstanding magnetic resonance imaging (MRI) response. Here we further explored the origin of this unprecedented response and expanded the choice of potential MRI probes by preparing the free acid version of the initial MRI sensor. We report a detailed investigation of the 1H NMR dispersion, 17O NMR, and isothermal titration calorimetry properties of these two MRI probes upon interaction with Zn2+. The performed experiments confirm selective interaction of the MRI probes and target metal cation, which causes substantial changes in the coordination sphere of the paramagnetic center. It also evidenced some aggregation, which enhances the relaxivity response. Interestingly, conversion of the methyl ester to the free carboxylic acid of the tyrosine moiety changes the nature of the aggregates and leads to a smaller relaxivity response. The probes interact with human serum albumin (HSA) in the absence of Zn2+, which leads to a possible modification of the coordination sphere of Gd3+ or a substantial change in the exchange rate of second-sphere water molecules. In the presence of Zn2+, the interaction with HSA is very weak, demonstrating the importance of the Zn2+ coordination sphere in the behavior of these systems.

Doxorubicin-Sensitized Luminescence of NIR-Emitting Ytterbium Liposomes: Towards Direct Monitoring of Drug Release.

Drug-loaded liposomes are typical examples of nanomedicines. We show here that doxorubicin, the anti-cancer agent in the liposomal drug Doxil, can sensitize Ytterbium (Yb3+ ) and generate its near-infrared (NIR) emission. When doxorubicin and amphiphilic Yb3+ chelates are incorporated into liposomes, the sensitized emission of Yb3+ is dependent on the integrity of the particles, which can be used to monitor drug release. We also established the first demonstration that the NIR Yb3+ emission signal is observable in living mice following intratumoral injection of the Yb3+ -doxorubicin-liposomes, using a commercial macroscopic setup equipped with a NIR camera.

Lanthanide DO3A-Complexes Bearing Peptide Substrates: The Effect of Peptidic Side Chains on Metal Coordination and Relaxivity.

Two DO3A-type ligands conjugated to substrates of urokinase (L3) and caspase-3 (L4) via a propyl-amide linker were synthesized and their lanthanide(III) (Ln3+) complexes studied. A model compound without peptide substrate (L2) and an amine derivative ligand mimicking the state after enzymatic cleavage (L1) were also prepared. Proton Nuclear Magnetic Relaxation Dispersion (NMRD) profiles recorded on the gadolinium(III) (Gd3+) complexes, complemented with the assessment of hydration numbers via luminescence lifetime measurements on the Eu3+ analogues, allowed us to characterize the lanthanide coordination sphere in the chelates. These data suggest that the potential donor groups of the peptide side chains (carboxylate, amine) interfere in metal coordination, leading to non-hydrated LnL3 and LnL4 complexes. Nevertheless, GdL3 and GdL4 retain a relatively high relaxivity due to an important second-sphere contribution generated by the strongly hydrophilic peptide chain. Weak PARACEST effects are detected for the amine-derivative EuL1 and NdL1 chelates. Unfortunately, the GdL3 and GdL4 complexes are not significantly converted by the enzymes. The lack of enzymatic recognition of these complexes can likely be explained by the participation of donor groups from the peptide side chain in metal coordination.

Metal-based environment-sensitive MRI contrast agents.

Interactions of paramagnetic metal complexes with their biological environment can modulate their magnetic resonance imaging (MRI) contrast-enhancing properties in different ways, and this has been widely exploited to create responsive probes that can provide biochemical information. We survey progress in two rapidly growing areas: the MRI detection of biologically important metal ions, such as calcium, zinc, and copper, and the use of transition metal complexes as smart MRI agents. In both fields, new imaging technologies, which take advantage of other nuclei (19F) and/or paramagnetic contact shift effects, emerge beyond classical, relaxation-based applications. Most importantly, in vivo imaging is gaining ground, and the promise of molecular MRI is becoming reality, at least for preclinical research.

From Zn(II) to Cu(II) Detection by MRI Using Metal-Based Probes: Current Progress and Challenges.

Zinc and copper are essential cations involved in numerous biological processes, and variations in their concentrations can cause diseases such as neurodegenerative diseases, diabetes and cancers. Hence, detection and quantification of these cations are of utmost importance for the early diagnosis of disease. Magnetic resonance imaging (MRI) responsive contrast agents (mainly Lanthanide(+III) complexes), relying on a change in the state of the MRI active part upon interaction with the cation of interest, e.g., switch ON/OFF or vice versa, have been successfully utilized to detect Zn2+ and are now being developed to detect Cu2+. These paramagnetic probes mainly exploit the relaxation-based properties (T1-based contrast agents), but also the paramagnetic induced hyperfine shift properties (paraCEST and parashift probes) of the contrast agents. The challenges encountered going from Zn2+ to Cu2+ detection will be stressed and discussed herein, mainly involving the selectivity of the probes for the cation to detect and their responsivity at physiologically relevant concentrations. Depending on the response mechanism, the use of fast-field cycling MRI seems promising to increase the detection field while keeping a good response. In vivo applications of cation responsive MRI probes are only in their infancy and the recent developments will be described, along with the associated quantification problems. In the case of relaxation agents, the presence of another method of local quantification, e.g., synchrotron X-Ray fluorescence, single-photon emission computed tomography (SPECT) or positron emission tomography (PET) techniques, or 19F MRI is required, each of which has its own advantages and disadvantages.

Synthesis and In Vitro Studies of a Gd(DOTA)-Porphyrin Conjugate for Combined MRI and Photodynamic Treatment.

With the aim of developing new molecular theranostic agents, a π-extended Zn(II) porphyrin as photosensitizer for photodynamic therapy (PDT) linked to two GdDOTA-type complexes for magnetic resonance imaging (MRI) detection was synthesized. The relaxivity studies revealed a much higher relaxivity value per Gd ion for this medium sized molecule (19.32 mM-1 s-1 at 20 MHz and 298 K) compared to clinical contrast agents-a value which strongly increases in the presence of bovine serum albumin, reaching 25.22 mM-1 s-1. Moreover, the photophysical studies showed the strong ability of the molecule to absorb light in the deep red (670 nm, ε ≈ 60000 M-1 cm-1) and in the near-infrared following two-photon excitation (920 nm, σ2 ≈ 650 GM). The conjugate is also able to generate singlet oxygen, with a quantum yield of 0.58 in DMSO. Promising results were obtained in cellular studies, demonstrating that the conjugate is internalized in HeLa cells at micromolar concentration and leads to 70% of cell death following 30 min irradiation at 660 nm. These results confirm the potential of the designed molecule as an imaging and therapeutic agent.

Aza-BODIPY Platform: Toward an Efficient Water-Soluble Bimodal Imaging Probe for MRI and Near-Infrared Fluorescence.

In this study, an original aza-BODIPY system comprising two Gd3+ complexes has been designed and synthesized for magnetic resonance imaging/optical imaging applications, by functionalization of the boron center. This strategy enabled the obtainment of a positively charged bimodal probe, which displays an increased water solubility, optimized photophysical properties in the near-infrared region, and very promising relaxometric properties. The absorption and emission wavelengths are 705 and 741 nm, respectively, with a quantum yield of around 10% in aqueous media. Moreover, the system does not produce singlet oxygen upon excitation, which would be toxic for tissues. The relaxivity obtained is high at intermediate fields (16.1 mM-1 s-1 at 20 MHz and 310 K) and competes with that of bigger or more rigid systems. A full relaxometric and 17O NMR study and fitting of the data using the Lipari-Szabo approach showed that this high relaxivity can be explained by the size of the system and the presence of some small aggregates. These optimized photophysical and relaxometric properties highlight the potential use of such systems for future bimodal imaging studies.

Toward MRI and Optical Detection of Zwitterionic Neurotransmitters: Near-Infrared Luminescent and Magnetic Properties of Macrocyclic Lanthanide(III) Complexes Appended with a Crown Ether and a Benzophenone Chromophore.

Thanks to their versatile magnetic and luminescence features, lanthanide complexes have gained a central position in biomedical imaging as magnetic resonance imaging (MRI) contrast agents and optical imaging probes. In addition, appropriate chemical design allows modification of the magnetic relaxation properties of GdIII complexes and the optical properties of visible- or near-infrared (NIR)-emitting lanthanide chelates upon interaction with various biomarkers, which makes them ideal candidates for the creation of responsive agents. In this Forum Article, we demonstrate such design principles as well as the difficulties encountered in the context of neurotransmitter (NT) detection. Lanthanide(III) complexes of a macrocyclic ligand incorporating a benzophenone chromophore and a monoazacrown ether (LnL3) have been synthesized as responsive probes to monitor amino acid NTs either in MRI (Ln = Gd) or in NIR optical detection (Ln = Nd or Yb). The parameters characterizing the water exchange and rotational dynamics of the gadolinium(III) complex were assessed by 17O NMR and 1H NMRD. In the presence of zwitterionic NTs, the inner-sphere water molecule is replaced by the carboxylate function of the NTs in the gadolinium(III) complex, leading to a decrease of the longitudinal relaxivity from 6.7 to 2-2.5 mM-1 s-1 (300 MHz and 37 °C). The apparent affinity constants range from Ka = 35 for γ-aminobutyric acid (GABA) to 80 M-1 for glycine and glutamate, and there is no selectivity with respect to hydrogen carbonate (Ka = 232; pH 7.4). The gadolinium(III) complex interacts with human serum albumin (HSA), resulting in a 60% increase in the relaxivity (20 MHz, 37 °C) in the absence of NTs. The HSA-bound complex, however, was revealed to be less responsive to NTs because of displacement of the GdIII-bound water by HSA, which was confirmed by the hydration number calculated from luminescence lifetimes of the HSA-bound europium(III) complex. The creation of an imaging agent suitable for NIR detection of NTs for an enhanced sensitivity in biological systems using the benzophenone (BP) moiety as the sensitizer of lanthanide luminescence was also attempted. Upon excitation at 300 nm of the BP chromophore in aqueous solutions of NdL3 and YbL3, characteristic NIR emissions of NdIII and YbIII were observed because of 4F3/2 → 4IJ (J = 9/2-13/2) and 2F5/2 → 2F7/2 transitions, respectively, indicating that this chromophore is a suitable antenna. Despite these promising results, luminescence titrations of NdIII and YbIII complexes with NTs were not conclusive because of chemical conversion of the ligand triggered by light, preventing quantitative analysis. The observed photochemical reaction of the ligand is strongly dependent on the nature of the lanthanide chelated; it is considerably slowed down in the presence of NdIII and EuIII.

High-Field Detection of Biomarkers with Fast Field-Cycling MRI: The Example of Zinc Sensing.

Many smart magnetic resonance imaging (MRI) probes provide response to a biomarker based on modulation of their rotational correlation time. The magnitude of such MRI signal changes is highly dependent on the magnetic field and the response decreases dramatically at high fields (>2 T). To overcome the loss of efficiency of responsive probes at high field, with fast-field cycling magnetic resonance imaging (FFC-MRI) we exploit field-dependent information rather than the absolute difference in the relaxation rate measured in the absence and in the presence of the biomarker at a given imaging field. We report here the application of fast field-cycling techniques combined with the use of a molecular probe for the detection of Zn2+ to achieve 166 % MRI signal enhancement at 3 T, whereas the same agent provides no detectable response using conventional MRI. This approach can be generalized to any biomarker provided the detection is based on variation of the rotational motion of the probe.

A Porphyrin Dimer-GdDOTA Conjugate as a Theranostic Agent for One- and Two-Photon Photodynamic Therapy and MRI.

A molecular theranostic agent designed for photodynamic therapy (PDT) treatment in the near-infrared and for imaging tissue tumors with magnetic resonance imaging (MRI) is reported. It consists of a linear π-conjugated Zn(II) porphyrin dimer linked at each extremity to a GdDOTA-type complex. This agent has shown very promising potential for PDT applications with good singlet oxygen generation in DMSO and high linear absorption in the near-infrared (λmax = 746 nm, ε ≈ 105 M-1 cm-1). Moreover, this molecule has a propensity for two-photon excited PDT with high two-photon cross sections (∼8000 GM in 880-930 nm range), which should allow for deeper tumor treatments and higher spatial precision as compared to conventional one-photon PDT. Regarding the MRI contrast agent properties, the molecule has shown superior relaxivity (14.4 mM-1 s-1 at 40 MHz, 298 K) in comparison to clinical contrast agents and the ability to be internalized in cells, thanks to its amphiphilic character. Irradiation of HeLa cells using either one-photon (740 nm) or two-photon excitation (910 nm) has led in both cases to important cell death.

MRI and luminescence detection of Zn2+ with a lanthanide complex-zinc finger peptide conjugate.

A bioinspired probe based on a zinc finger peptide functionalized by a lanthanide(iii)-DOTA monoamide complex turns out to be active for both luminescence and MRI detection of Zn2+, depending on the lanthanide cation. A mechanism for MRI-based detection is proposed.

A cocktail of 165Er(iii) and Gd(iii) complexes for quantitative detection of zinc using SPECT and MRI.

We propose quantitative assessment of zinc by combining nuclear and MR imaging. We use a cocktail of a Gd3+-complex providing a Zn2+-dependent MRI response and its 165Er3+ analogue allowing for concentration assessment. 165Er is readily obtained in a cyclotron and purified, which is indispensable for successful quantification of metal ions.

Surface PEG Grafting Density Determines Magnetic Relaxation Properties of Gd-Loaded Porous Nanoparticles for MR Imaging Applications.

Surface PEGylation of nanoparticles designed for biomedical applications is a common and straightforward way to stabilize the materials for in vivo administration and to increase their circulation time. This strategy becomes less trivial when MRI active porous nanomaterials are concerned as their function relies on water/proton-exchange between the pores and bulk water. Here we present a comprehensive study on the effects of PEGylation on the relaxometric properties of nanozeolite LTL (dimensions of 20 × 40 nm) ion-exchanged with paramagnetic GdIII ions. We evidence that as long as the surface grafting density of the PEG chains does not exceed the "mushroom" regime (conjugation of up to 6.2 wt % of PEG), Gd-LTL retains a remarkable longitudinal relaxivity (38 s-1 mM-1 at 7 T and 25 °C) as well as the pH-dependence of the longitudinal and transverse relaxation times. At higher PEG content, the more compact PEG layer (brush regime) limits proton/water diffusion and exchange between the interior of LTL and the bulk, with detrimental consequences on relaxivity. Furthermore, PEGylation of Gd-LTL dramatically decreases the leakage of toxic GdIII ions in biological media and in the presence of competing anions, which together with minimal cytotoxicity renders these materials promising probes for MRI applications.

Synthetic water soluble di-/tritopic molecular receptors exhibiting Ca2+/Mg2+ exchange.

Structural integration of two synthetic water soluble receptors for Ca2+ and Mg2+, namely 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and o-aminophenol-N,N,O-triacetic acid (APTRA), respectively, gave novel di- and tritopic ionophores (1 and 2). As Mg2+ and Ca2+ cannot be simultaneously complexed by the receptors, allosteric control of complexation results. Potentiometric measurements established stepwise protonation constants and showed high affinity for Ca2+ (log K = 6.08 and 8.70 for 1 and 2, respectively) and an excellent selectivity over Mg2+ (log K = 3.70 and 5.60 for 1 and 2, respectively), which is compatible with magnesium-calcium ion exchange. While ion-exchange of a single Mg2+ for a single Ca2+ is possible in both 1 and 2, the simultaneous binding of two Mg2+ by 2 appears prohibitive for replacement of these two ions by a single Ca2+. Ion-binding and exchange was further rationalized by DFT calculations.

Proton Exchange in a Paramagnetic Chemical Exchange Saturation Transfer Agent from Experimental Studies and ab Initio Metadynamics Simulation.

The proton-exchange process between water and a carbamate has been studied experimentally and theoretically in a lanthanide-based paramagnetic chemical exchange saturation transfer agent endowed with potential multimodality detection capabilities (optical imaging, or T1 MRI for the Gd(III) analogue). In addition to an in-depth structural analysis by a combined approach (using X-ray crystallography, NMR, and molecular dynamics), our ab initio simulation in aqueous solution sheds light on the reaction mechanism for this proton exchange, which involves structural Grotthuss diffusion.