Introduction

The complexity of biology attracts scientists and clinicians from a wide range of fields. Indeed, optical based dyes have been widely developed and used to study such complexity in a multicolor imaging fashion. However, their light signal source calls for alternatives that could be performed on live intact subjects. In MRI, both diaCEST¹ and paraCEST² based probes have been designed and used for multiplexed imaging mimicking optical imaging capabilities exploiting the Δw of the exchangeable proton in the designed agents. Here, we show that performing the CEST approach in ¹⁹F-MRI framework allows obtaining “multicolor” imaging features of multiple targets using single ¹⁹F-agent. Based on different binding kinetics in host:guest molecular systems and distinct Δws obtained in ¹⁹F-MR, a novel platform for multiplexed MR imaging is presented.

Methods

All NMR and MRI experiments were performed on 9.4T scanners (Bruker, Germany). ¹⁹F-CEST NMR data were acquired as follows: A pre-saturation pulse with a length (t_sat) of 3 sec was applied prior to the 90⁰ RF pulse. The saturation pulse strength was set to 150Hz for all samples. The frequency of the pre-saturation pulse was swept from Δω=+6.1ppm to Δω=-6.1ppm in 100Hz=0.26ppm steps relative to the resonance of the free ¹⁹F-agent (i.e., fluoroxene, set to 0ppm for convenience). For each frequency offset (MΔωi), the following parameters were used: NS=8, TR=15. For ¹H-MRI, a FLASH sequence was used with the following parameters TR/TE=100/6 ms; 2 mm slice; FOV=2.56×2.56 cm²; matrix size=128×128. For ¹⁹F-MRI, RARE sequence was used TR/TE=6000/3.64 ms; 10 mm slice thickness; FOV=3.2×3.2 cm²; matrix size=64×64. For the ¹⁹F-CEST MRI the frequency of the pre-saturation pulse (B1=3.6 μT/ 3000 ms) was swept from Δω=+5ppm to Δω=-5ppm in 100Hz=0.26ppm steps.

Results and discussion

Recently, the use of the CEST approach in ¹⁹F-NMR framework for studying the binding kinetics in several host:guest supramolecular systems was shown^3-4. In this approach, the magnetization of a low concentration of host:guest complex is transferred to the signal of the free guest through a dynamic exchange process. In the present study, two supramolecular systems composed of molecular hosts (either cucurbit[n]uril, CB[7]; or octa acid, OA, Fig. 1a) and ¹⁹F-guest (fluoroxene, Fig. 1a right panel) were used. Following calculation of the relaxation parameters of the fluorinated agent fluoroxene (T₁=4.34 sec and T₂=3.33 sec) the ¹⁹F-CEST characteristics of CB[7]:fluoroxene and OA:fluoroxene systems in PBS were studied and summarized in Fig. 1b. Obviously, without a molecular host (CB[7] or OA) in the sample, no ¹⁹F-CEST effect was observed (Fig. 1b, red curve). Interestingly, when low concentration (50 μM) of molecular host (CB[7] or OA) was added into the studied solution a large CEST effect was obtained (45-50% signal change for 1:100 host:guest ratio) for both studied systems. Importantly, while for CB[7]:fluoroxene system the obtained ¹⁹F-CEST effect was at Δw of +1.6 ppm (Fig. 1b, purple curve), the effect for OA:fluoroxene system was opposite, at Δw of -2.0 ppm (Fig. 1b, green curve). This observation, of Δw-CEST dependence, encouraged us to use this platform for multiplexed ¹⁹F-MRI. A phantom consisting of 4 different host:guest pairs (1:100 molar ratio, 50 μM of host and 5 mM ¹⁹F-guest) in PBS were prepared as shown in Fig. 2a) – CB[6]:fluoroxene, CB[7]:fluoroxene, β-cyclodextrin:fluoroxene and OA:fluoroxene. As expected, ¹H-MRI and ¹⁹F-MRI did not reveal any difference between the samples in the phantom. However, by performing the ¹⁹F-CEST MRI experiment a multicolor imaging characteristic was obtained. While the pre-saturation pulse was applied at Δw=+1.6 ppm, the CEST effect was obtained only from the CB[7] (50 μM) containing tube. Contrary, while the pre-saturation pulse was applied at Δw=-2.0 ppm, the CEST effect was obtained only from the OA (50 μM) containing tube. These observations are shown as pseudo-multicolor imaging map overlaid on ¹H-MR image demonstrating the capabilities of using the proposed platform for multiplexed MR imaging. It is important to mention that the low concentrations of molecular targets used here (50 μM of CB[7] or OA) can not be directly detected with ¹⁹F-MRI. However, the use of ¹⁹F-CEST allows the observation of these targets (50 μM) with the sensitivity of the free ¹⁹F-agent (5 mM).

Conclusion

Conclusion

Conclusion

We have demonstrated the feasibility of applying the ¹⁹F-CEST methodology on a variety of host:guest systems to amplify ¹⁹F-MR signals of low concentration targets (~mM levels of molecular host). Capitalizing on the different Δw obtained for the ¹⁹F-guest upon encapsulation, a multicolor ¹⁹F-CEST MRI can be obtained. By targeting the molecular hosts (CB[7] or OA) to multiple low-concentration targets the proposed platform has the potentiality to monitor such targets, simultaneously, using single ¹⁹F-probe.

Acknowledgements

No acknowledgement found.