Paramagnetic fluorinated nanoemulsions for sensitive cellular fluorine-19 magnetic resonance imaging

Abstract

Fluorine-19 magnetic resonance imaging ((19)F MRI) probes enable quantitative in vivo detection of cell therapies and inflammatory cells. Here, we describe the formulation of perfluorocarbon-based nanoemulsions with improved sensitivity for cellular MRI. Reduction of the (19)F spin-lattice relaxation time (T1) enables rapid imaging and an improved signal-to-noise ratio, thereby improving cell detection sensitivity. We synthesized metal-binding β-diketones conjugated to linear perfluoropolyether (PFPE), formulated these fluorinated ligands as aqueous nanoemulsions, and then metallated them with various transition and lanthanide ions in the fluorous phase. Iron(III) tris-β-diketonate ('FETRIS') nanoemulsions with PFPE have low cytotoxicity (<20%) and superior MRI properties. Moreover, the (19)F T1 can readily be reduced by an order of magnitude and tuned by stoichiometric modulation of the iron concentration. The resulting (19)F MRI detection sensitivity is enhanced by three- to fivefold over previously used tracers at 11.7 T, and is predicted to increase by at least eightfold at the clinical field strength of 3 T.

Figure 1. Comparison of iron and gadolinium diketonates (H-fod) as ¹⁹F relaxation agents for PFPE

The relaxometry results (9.4 T) are displayed for PFPE emulsions (120 g/L PFPE) containing H-fod (2.8 mM) 24 hours after the addition of 0.7 mM metal ions. R₁ and R₂ values are reported for the main PFPE peak at −91.4 ppm. The results show that Fe³⁺ is a more effective R₁ agent than Gd³⁺.

Figure 2. Preparation and characterization of metal-binding nanoemulsions for ¹⁹F MRI

a, Synthesis of metal-binding fluorinated diketones (FDK) from PFPE-OMe (denoted as R_FCO₂Me). b, Structures of fluorocarbons used for ¹⁹F MRI. c, Composition and preparation of various metal-binding (A, B, D, F, G) and control (C, E) fluorocarbon nanoemulsions. d, ¹⁹F NMR spectra (11.7 T) of emulsions A–C (4.5 g/L ¹⁹F, 90% D₂O). Signals from terminal CF₂ of diketone ligands are well separated from other peaks and are used to determine ligand concentration. The peak at −76 ppm is the reference (CF₃CO₂Na, TFA). e, Addition of aqueous metal chlorides to FDK emulsions yields metalated emulsions; Ar = pAn. f, Absorption spectra of metal-binding emulsion B (70 µM diketone, 0.09 g/L ¹⁹F) (color) and control emulsion C (0.09 g/L) (---) in the presence of Fe³⁺. Increasing [Fe³⁺] causes the appearance of ferric tris-diketonate charge transfer bands at 395 and 500 nm that grow linearly in intensity until the ca. 3:1 ligand:Fe ratio is reached at 25 µM Fe³⁺.

Figure 3. Fluorine-19 relaxometry of metalated PFPE emulsions

a, R₁ and ¹⁹F NMR spectra of FETRIS nanoemulsion (4.5 g/L ¹⁹F, 3.5 mM diketone) in the presence of 0.5 mM metal ions, 15 mM HEPES, and at pH 7.4. The peaks from different ¹⁹F spectra are scaled to the same absolute intensity. b, Relaxometric analysis of Fe³⁺ and Gd³⁺ binding capacity. Shown are measurements of R₁ for both PFPE (fluorous phase) and trifluoroacetate reference (TFA) added to the aqueous phase. c, Magnetic field dependence at T = 295 K and d, temperature (B₀ = 9.4 T) dependence of observed relaxation rates R₁ (●) and R₂ (x) in FETRIS nanoemulsion (22.5 g/L ¹⁹F, 17.5 mM diketone, 2.8 mM Fe³⁺) and predicted R₁ (—) values using Eqs. S1–S4. Predicted R₁ values represent best fit to SBM equations, with r = 1.19 nm, τ_F (295 K) = 0.80 ns, τ_v (295 K) = 3.59 ps, the Arrhenius temperature dependence with activation energies of 3.6 kcal/mol for τ_F and 4.5 kcal/mol for τ_v. The diamagnetic contributions to R₁ are presumed to be negligible and Δ fixed at 0.2 cm⁻¹. R₁ values increase, while R₂ values decrease, at lower magnetic field strengths, suggesting that there will be no degradation of SNR at clinical fields due to line broadening.

Figure 4. Relaxometry stability of FETRIS nanoemulsions in the presence of competing aqueous ligand

Nanoemulsion B and F, both metalated with 0.7 mM Fe³⁺, were treated at 37 °C with 75 mM EDTA dissolved in aqueous phase. Shown are R₁ values of PFPE () in nanoemulsion B, and values for blend nanoemulsion F, including PFPE components () and the CF₃ signal of PFOB (). A slight decrease over time is observed, as slow Fe³⁺ efflux occurs from the fluorous phase and irreversibly binds to EDTA. Error bars are standard deviations from three independent replicates.

Figure 5. Cell labeling with FETRIS nanoemulsion

Cells (GL261) were labeled in culture using FETRIS nanoemulsion. a, Cell viability. b, Cell uptake of FETRIS as measured by ¹⁹F NMR. c, Correlation of uptake determined by ¹⁹F NMR with optical absorbance of cell lysate at 390 nm due to FETRIS. Error bars are standard deviations from three independent replicates.

Figure 6. MRI of FETRIS nanoemulsion

a. Phantom comprised of two agarose-embedded NMR tubes containing FETRIS nanoemulsion (4.5 g/L ¹⁹F) with 0.5 mM Fe³⁺ (R₁/R₂ = 32.5/170 s⁻¹) and nanoemulsion without metal (R₁/R₂ 2.2/3.7 s⁻¹), denoted +Fe and −Fe, respectively. The top panel shows unthresholded ¹⁹F images, and below, the ¹⁹F image is thresholded, rendered in hot-iron pseudo-color (scale bar), and overlaid onto the grayscale ¹H image. The ¹⁹F/¹H MRI data were acquired using a GRE sequence. b, Displays mouse GL261 glioma cells (5×10⁶) labeled with FETRIS nanoemulsion ex vivo and injected subcutaneously into mouse flank. The ¹⁹F data is rendered in pseudo-color and placed on a grayscale slice from the ¹H data. After 24 hours, mice were imaged, and a cell ‘hot-spot’ is seen on the right flank in the axial view. Cells labeled with metal-free nanoemulsion and injected on the contralateral side could not be detected. Asterisk is adjacent chemical shift displacement artifact from hyperintense subcutaneous fat at 11.7T. The ¹⁹F and ¹H images were acquired using ZTE and GRE pulse sequences, respectively. For display, a co-registered 2D GRE slice was embedded into a 3D rendering of the ¹⁹F data.