Introduction

Fluorine-19 MRI is a cellular imaging approach enabling quantitative ‘hot-spot’ imaging with no background signal. The utility of ¹⁹F-MRI to detect inflammation and cell therapy in vivo could be expanded by improving the intrinsic sensitivity of the probe by molecular design. We describe a fluorinated metal chelate based on a salicylidene-tris(aminomethyl)ethane core, with solubility in perfluorocarbon (PFC) oils, and a potent accelerator of the 19F longitudinal relaxation time (T₁) via the intermolecular paramagnetic relaxation enhancement mechanism due to the paramagnetic centers. Shortening T₁ can increase the ¹⁹F image sensitivity per time and decrease the minimum number of detectable cells with signal averaging. We dissolved Fe³⁺-chelate with perfluorooctyl-bromide (PFOB) to formulate a stable paramagnetic nanoemulsion imaging probe (P-PFOB) and assessed its biocompatibility in macrophages in vitro. From empirical relaxivities of P-PFOB, we performed signal-to-noise (SNR) modeling of P-PFOB. We demonstrate the utility of this paramagnetic nanoemulsion as an in vivo MRI probe for detecting inflammation macrophages in mice.

Methods

We designed and synthesized a stable hexadentate chelating agent for iron (III). We used the condensation between tris-1,1,1-(aminomethyl)ethane (TAME) and salicylaldehyde (SAL) to form the tripodal salicylidene-tris(aminomethyl)ethane chelating agent (SALTAME). The structure of this complex was confirmed by NMR and x-ray crystallography. We added the chelating agent to PFOB to form paramagnetic PFOB (P-PFOB) nanoemulsion using microfluidization. To stabilize the nanoemulsion, we used egg yolk phospholipid surfactant. Nanoemulsion particle size was measured by dynamic light scattering (DLS). Relaxivity (R₁ and R₂) ¹⁹F NMR measurements (9.4T and 3T) and in vitro cell apoptosis assays where used to characterize the nanoemulsion. For in vivo studies, inflammation was induced in a C57BL/6J murine model by implanting a subcutaneous plug of Matrigel mixed with lipopolysaccharide (LPS) in the neck. P-PFOB nanoemulsion was subsequently injected intravenously, and 11.7T MRI data were acquired 24 h later. A ¹⁹F 2D chemical shift imaging (CSI) sequence with: 134 averages, TR=13.3 ms, TE=0.53 ms, and matrix size 32×32. For T₂*-weighted 1H, TR/TE=550/14 ms and matrix size 128×96.

Results

We purified four isomers of SALTAME, elucidated structures using x-ray scattering (Fig. 1a) and NMR, and identified a single isomer with high PFOB solubility. Using the soluble isomer, we screened the impact of cations bound to SALTAME and dissolved in PFOB via NMR relaxometry; of the cations tested (V³⁺, Cr³⁺, Mn²⁺, Fe³⁺, Co²⁺, Cu²⁺, Ni²⁺, Zn²⁺, Ga³⁺), only Mn⁴⁺, Fe³⁺, Co³⁺, and Ga³⁺ formed stable chelates with SALTAME. After emulsification of P-PFOB, average nanoemulsion particle size was ~162 nm by DLS. Consistent with prior studies,¹ one of the cations only Fe³⁺ yielded superior T₁ shortening with modest line broadening. The ¹⁹F relaxation rates of P-PFOB was evaluated as a function of [Fe³⁺] at 3T to obtain relaxivities r₁ and r₂ of 0.56 s^-1mM^-1 and 1.67 s^-1mM^-1, respectively, compared to 0.50 s^-1mM^-1 and 1.07 s^-1mM^-1 values of r₁ and r₂ at 9.4T, respectively. For neat PFOB, R₁/R₂ values are 0.79 s^-1/3.5 s^-1 and 1.4 s^-1/2.2 s^-1 at 3T and 9.4T, respectively. We also evaluated the iron-binding stability of P-PFOB nanoemulsion by adding a competing chelate (EDTA), and stable relaxometry parameters were observed over 20 days. SNR modeling (Fig. 1b) of P-PFOB shows that sensitivity enhancement of nearly 4-fold is feasible at clinical magnetic field strengths using a short-TE gradient-echo pulse sequence. To demonstrate the feasibility of detecting macrophage inflammation with P-PFOB nanoemulsion in vivo, in situ macrophage labeling² in a LPS-Matrigel mouse model. A bolus of P-PFOB nanoemulsion (200 μl inoculant, [Fe3+-SALTAME]=20 mM in PFOB oil phase) was injected intravenously. Mice were imaged 24 hours later to allow for nanoemulsion uptake by monocytes/macrophages in situ. Scans were performed at 11.7T, and ¹⁹F images were acquired using a 2D CSI sequence along with anatomical ¹H images (Fig. 1c). Matrigel plug appears as a hyperintense, subcutaneous structure in the dorsal region of the ¹H image (Fig. 1c). The ¹⁹F signal (macrophage) is seen with the Matrigel plug and in anterior neck lymph node (Fig. 1c).

Conclusion

We describe SALTAME, a stable hexadentate chelating agent for iron (III). We used this moiety to formulate a nanoemulsion MRI probe that can be used for ‘hot-spot’ detection in vivo. Incorporation of iron-bound SALTAME into the fluorous phase causes a profound reduction of the ¹⁹F T₁ value and only mild line broadening, thus offering improved sensitivity of ¹⁹F MRI due to increased signal averaging and/or reduced MRI scan time. This probe has the potential to enable non-invasive quantification of inflammation and therapeutic cell delivery and aid in the monitoring of therapeutic test articles.

Acknowledgements

Funding for ETA was provided by National Institutes of Health (NIH) grants R01-EB017271, R01-EB024015, R01 CA139579 and the California Institute for Regenerative Medicine LA1-C12-06919.