Quantitative zinc determination by nuclear and MR imaging using two Ln ³⁺ complexes, including purified ¹⁶⁵ Er ³⁺ , indispensable for metal ion quantification.

Lanthanide(III) (Ln³⁺) complexes have desirable photophysical properties for optical bioimaging. However, despite their advantages over organic dyes, their use for microscopy imaging is limited by the high-energy UV excitation they require and their poor ability to cross the cell membrane and reach the cytosol. Here we describe a novel family of lanthanide-based luminescent probes, termed dTAT[Ln·L], based on (i) a DOTA-like chelator with a picolinate moiety, (ii) a two-photon absorbing antenna to shift the excitation to the near infrared and (ii) a dimeric TAT cell-penetrating peptide for cytosolic delivery. Several Tb³⁺ and Eu³⁺ probes were prepared and characterized. Two-photon microscopy of live cells was attempted using a commercial microscope with the three probes showing the highest quantum yields (>0.15). A diffuse Ln³⁺ emission was detected in most cells, which is characteristic of cytosolic delivery of the Ln³⁺ complex. The cytotoxicity of these three probes was evaluated and the IC₅₀ ranged from 7 μM to >50 μM. The addition of a single positive or negative charge to the antenna of the most cytotoxic compound was sufficient to lower significantly or suppress its toxicity under the conditions used for two-photon microscopy. Therefore, the design reported here provides excellent lanthanide-based probes for two-photon microscopy of living cells.

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.

Amino-terminal Cu^II and Ni^II (ATCUN) binding sequences are widespread in the biological world. Here, we report on the study of eight ATCUN peptides aimed at targeting copper ions and stopping the associated formation of reactive oxygen species (ROS). This study was actually more focused on Cu(Aβ)-induced ROS production in which the Aβ peptide is the “villain” linked to Alzheimer’s disease. The full characterization of Cu^II binding to the ATCUN peptides, the Cu^II extraction from Cu^II(Aβ), and the ability of the peptides to prevent and/or stop ROS formation are described in the relevant biological conditions. We highlighted in this research that all the ATCUN motifs studied formed the same thermodynamic complex but that the addition of a second histidine in position 1 or 2 allowed for an improvement in the Cu^II uptake kinetics. This kinetic rate was directly related to the ability of the peptide to stop the Cu^II(Aβ)-induced production of ROS, with the most efficient motifs being HWHG and HGHW.

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.

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 K_Gd = 16.27) versus Zn²⁺ (log K_Zn = 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 (K_D = 170 μM versus K_DZn = 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.