Visualization of distinct cell populations in vivo is a formidable challenge in biomedical sciences. Clinical translation of cutting-edge therapies that involve administration of live immunotherapeutic or stem cells can benefit from understanding the fate of injected cells in vivo. Magnetic resonance imaging (MRI) is emerging as a clinically acceptable method for non-invasive, longitudinal cell tracking. Fluorine-19 MRI (¹⁹F MRI) involves the use of a non-radioactive ¹⁹F label in the form of biologically inert, cytocompatible perfluorocarbons (PFC). Immediately prior to injection, cells of interest are labeled ex vivo with PFC nanoemulsions. ¹⁹F MRI yields background-free images of labeled cells in an anatomical context provided by conventional ¹H MRI. This technology has recently been used to detect immunotherapeutic dendritic cells delivered to colorectal adenocarcinoma patients.¹

In this study, we describe a new PFC material that contains chelated iron(III) in the form of ferric tris-β-diketonates (FETRIS), attached covalently to the termini of polyfluoropolyether (PFPE) molecules. FETRIS displays reduced ¹⁹F longitudinal relaxation time (T₁), the parameter that governs the data acquisition rate of MRI, in order to enhance sensitivity and versatility of ¹⁹F MRI cell tracking.

Relaxometry. PFPEs, covalently modified with metal-binding β-diketones, were formulated as nanoemulsions and metalated with metal chlorides in water. NMR relaxation rates (R₁=1/T₁ and R₂=1/T₂) were measured on a Jeol ACA-500 (11.7 T, pH 7.4).

In vitro ¹⁹F MRI. All images were acquired on Bruker 11.7 T Biospec using a ¹⁹F/¹H double tuned volume coil. The phantom was comprised of two 5 mm NMR tubes (PFC nanoemulsion without metal with R₁/R₂ = 2.2/3.7 s^–1 and FETRIS with R₁/R₂ = 32.5/170 s^–1) that were embedded in agarose. For ¹⁹F, a GRE pulse sequence was used with parameters TR/TE=15/0.83 ms, NA=256, FOV=4×4 cm², 64×64 matrix, 8 mm thick slices, FA=Ernst angle optimal for FETRIS sample, and acquisition time ~4 min. For ¹H, the GRE parameters were TR/TE 150/2 ms, NA=8, FOV=4×4 cm², 256×256 matrix, 2 mm slices. The ¹⁹F image data was rendered in hot-iron pseudo-color in ImageJ and overlaid onto grayscale ¹H image.

In vivo ¹⁹F MRI. Mouse GL261 glioma cells were labeled with FETRIS ex vivo to a level of ~10¹² F/cell. A second batch of cells was similarly labeled with unmetalated nanoemulsion. Cells (5×10⁶ in 0.4 mL Matrigel) were injected subcutaneously into flanks (left side, no metal; right side, FETRIS) in female syngeneic C57BL/6 mice (8–10 wks old, n=3). After 24 hrs, mice were imaged using 3D ZTE sequence with TR=4 ms, BW=40 kHz, acquisition window 0.8 ms, number of projections 13030, NA=26, FOV=6×6×6 cm³, matrix size 64×64×64, and acquisition time = 23 min. Proton data were acquired using a 2D spin-echo sequence with TR/TE=1500/14 ms, FOV=6×6 cm², and 256×256 matrix. ¹⁹F data were imported into Amira (FEI, Hillsboro, OR) and rendered in color and a grayscale slice from the ¹H data was embedded for anatomical context.

Stable nanoemulsion incorporating iron(III) in the fluorous phase (termed FETRIS) was synthesized. Paramagnetic relaxation enhancement by Fe³⁺ resulted in up to a 70-fold increase in ¹⁹F relaxation rate R₁ compared to non-metalated nanoemulsion tracers. Fe³⁺ was more effective than conventional R₁ enhancers Gd³⁺ and Mn²⁺ (Fig. 1) for same ion concentration. SNR of FETRIS phantom was ~5-fold higher than in identical sample without Fe³⁺ (Fig. 2a). We evaluated the new PFC nanoemulsions in labeled cells and tested the efficacy of these agents for MRI in mice bearing subcutaneous allografts of ¹⁹F-labeled GL261 cells. The ultralow ¹⁹F T₁ of these paramagnetic tracers enabled in vivo cell tracking using a rapid, acoustically silent, three-dimensional zero time-to-echo (ZTE) ¹⁹F MRI pulse sequence (Fig. 2b). We observed ¹⁹F signal in the right injected flank (FETRIS), but not on the left side (no metal).Gd³⁺ and Fe³⁺ are at the heart of T₁- and T₂-based ¹H contrast agents, respectively, but for ¹⁹F MRI, the roles of these metal ions are reversed. Fe³⁺ was the optimal T₁ enhancer for perfluorocarbons, while analogous gadolinium (and manganese) chelates caused severe line broadening, essentially becoming ¹⁹F T₂ agents. Combination of FETRIS with conventional ¹⁹F tracers with different relaxation properties enables contrast-based "multicolor" ¹⁹F MRI for detecting multiple cell populations in vivo, e.g. for monitoring the interaction of host immune cells with injected stem cells or cancer xenografts.We describe FETRIS, a new PFC material that contains chelated iron(III). FETRIS displays greatly reduced ¹⁹F T₁. Overall, the design of biocompatible, fast-T₁ ¹⁹F tracers can greatly boost the sensitivity of ¹⁹F MRI cell tracking, thereby reducing the barriers to widespread adoption of this powerful imaging technique.