Introduction:
Small molecular imaging probes for MRI applications are important set of sensors that were developed for mapping a variety of analytes ranging from neurotransmitters to metal ions. Nevertheless, their fast clearance from tissues frequently limits their detectability but also prevent the performance of longitudinal studies. Prolonged infusion strategies were found to be a solution for rapidly cleared responsive probes in in vivo studues^1-4 but other strategies for extending the tissue retention time of designed probes are still of need for desired applications. Here, we show the design of MRI probe, which form nano-assembly in live animal under physiological condition. A synthetic fluorinated-probe that can be used to detect labile Zn²⁺ in vitro by combining CEST and ¹⁹F-MRI (¹⁹F-iCEST)^5-8 was detectable in vivo even 21 hours after its injection allowing longitudinal in vivo MRI studies. This design strategy can be adopted to any MRI application where longitudinal in vivo imaging is needed.
Method:
A fluorinated-chelate that is based on a dipicolylamine motif, which specifically binds Zn²⁺, was synthesized followed by its coupling to a BTA (benzene-1,3,5-tricarboxamide) backbone to obtain the tripod compound 1. ¹⁹F-NMR and ¹⁹F-iCEST (B₁=2.3 µT/2 s) were performed on 9.4 T NMR at 25 ^oC and 37 ^oC. Nano-assembly formation was evaluated by dynamic light scattering (DLS),¹⁹F-NMR, diffusion NMR studies and by visualization at 25 ^oC and 37 ^oC. In vivo experiments were performed on 15.2 T MRI scanner after intracranial injection of 1 µL of compound 1 (75 mM in DMSO) to the CA3 region of the hippocampus in the mouse brain. In vivo ¹⁹F-NMR and ¹⁹F-MRI experiments were performed to study the sensor clearance rate from the live animal brain. For ¹⁹F-MRI experiment, ¹⁹F-RAREst sequence was used with the following parameters: TR/TE=4000/10.8 ms, RARE factor=16, FOV=22X22 mm² (32x32 matrix).
Results:
The nano-assembled Zn²⁺ responsive MRI probe compound 1 (Fig. 1a) with its ¹⁹F-dipicolylamine motifs as Zn²⁺ binding moieties was synthesized and characterized based on the hypothesis that tripod of this type tend to assemble to large architectures at elevated temperatures. Supramolecular structure formation of compound 1 at 37 ^oC in aqueous solution was confirmed by dynamic light scattering (DLS) measurements ¹⁹F-NMR and diffusion NMR. The DLS measurements showed the formation of 100 nm size assemblies only at 37 ^oC (Fig. 1b) and ¹⁹F-NMR showed an additional peak at elevated temperature, (upfield, Fig. 1c) that was assigned to the obtained large assemblies. The diffusion NMR studies confirmed that the additional upfield peak at the ¹⁹F-NMR is of the large assemblies showing x10 slower diffusion coefficient that is characteristic for large molecular entities (Fig. 1d). The formation of the large assemblies at 37 ^oC was observed by a naked-eye showing clear cloudiness of the heated solution (Fig. 1d, inset). The addition of Zn²⁺ to an aqueous solution of compound 1 resulted in an additional peak of Zn²⁺-1 complex in ¹⁹F-NMR spectrum (Δω=+5 ppm, Fig. 2a) and a characteristic ¹⁹F-iCEST spectrum showed a high CEST effect (Fig. 2a) from compound 1 in presence of labile Zn²⁺ at 37 ^oC revealing its potentiality to map the ion with MRI. Intracranially injection of compound 1 to a mouse brain was followed by in vivo ¹⁹F-MRS and ¹⁹F-MRI. The long retention time (>20 hours) of the injected probe was clearly evident by longitudinal in vivo ¹⁹F-MRS (Fig. 2b). In vivo ¹⁹F-MRI show the spatial distribution of compound 1 even 21 hours after its injection (Fig. 2c) confirming its prolonged retention time in the delivered region.
Conclusion:
We showed the design of MRI-responsive probe for Zn²⁺ sensing that forms nano-assemblies upon its injection to a live animal. We have demonstrated that the formed supramolecular structures is capable to detect Zn²⁺ when implementing the CEST principles in ¹⁹F-MRI (an approach termed ¹⁹F-iCEST). Moreover, we showed that upon its delivery, the ¹⁹F-MRI signal is preserved for more than 20 h after its injection. While the proposed approach was demonstrated here for a Zn²⁺ responsive probe that was designed for ¹⁹F-iCEST it can be generalized for the design of additional MRI responsive probes for applications that require long tissue-retention times for longitudinal in vivo studies.

Acknowledgements

No acknowledgement found.