Fluorine-19 MRI is an emerging cellular imaging approach, enabling lucid, quantitative “hot-spot” imaging with no background signal. The utility of 19F-MRI to detect inflammation and cell therapy products in vivo could be expanded by improving the intrinsic sensitivity of the probe by molecular design. We describe a 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). Shortening T1 can increase the 19F image sensitivity per time and decrease the minimum number of detectable cells. We used the condensation between the tripodal ligand tris-1,1,1-(aminomethyl)ethane and salicylaldehyde to form the salicylidene-tris(aminomethyl)ethane chelating agent (SALTAME). We purified four isomers of SALTAME, elucidated structures using X-ray scattering and NMR, and identified a single isomer with high PFC solubility. Mn4+, Fe3+, Co3+, and Ga3+ cations formed stable and separable chelates with SALTAME, but only Fe3+ yielded superior T1 shortening with modest line broadening at 3 and 9.4 T. We mixed Fe3+ chelate with perfluorooctyl bromide (PFOB) to formulate a stable paramagnetic nanoemulsion imaging probe and assessed its biocompatibility in macrophages in vitro using proliferation, cytotoxicity, and phenotypic cell assays. Signal-to-noise modeling of paramagnetic PFOB shows that sensitivity enhancement of nearly 4-fold is feasible at clinical magnetic field strengths using a 19F spin-density-weighted gradient-echo pulse sequence. We demonstrate the utility of this paramagnetic nanoemulsion as an in vivo MRI probe for detecting inflammation macrophages in mice. Overall, these paramagnetic PFC compounds represent a platform for the development of sensitive 19F probes.
A novel perfluoroalkyl-BINOL-based chiral diketone is found to be the first highly enantioselective fluorescent sensor in the fluorous phase. One enantiomer of a chiral amino alcohol or diamine at a concentration greater than 1 mM can cause an up to 1200-2000-fold fluorescent enhancement of the sensor (0.08 mM), while the other enantiomer gives only a 10-50-fold enhancement. The fluorous-phase-based sensor is found to enhance the reactivity of the previously reported fluorous insoluble sensor with amino alcohols and expand its chiral recognition ability. Dynamic light scattering studies show the formation of aggregates of very different particle sizes when two enantiomers of a substrate interact with the sensor in perfluorohexane (FC-12). This substantial difference enables easy discrimination of the enantiomers with UV-lamps or even the naked eye. NMR, IR, and mass spectroscopic studies indicate that the fluorescent enhancement and enantioselectivity should originate from the fluorous solvent-promoted nucleophilic addition of the amino alcohols to the carbonyl groups of the sensor.
Metrics & More Article RecommendationsCONSPECTUS: This Account summarizes recent advances in the chemistry of fluorocarbon nanoemulsion (FC NE) functionalization. We describe new families of fluorous molecules, such as chelators, fluorophores, and peptides, that are soluble in FC oils. These materials have helped transform the field of in vivo molecular imaging by enabling sensitive and cell-specific imaging using magnetic resonance imaging (MRI), positron emission tomography (PET), and fluorescence detection. FC emulsions, historically considered for artificial blood substitutes, are routinely used for ultrasound imaging in clinic and have a proven safety profile and a well-characterized biodistribution and pharmacokinetics. The inertness of fluorocarbons contributes to their low toxicity but makes functionalization difficult. The high electronegativity of fluorine imparts very low cohesive energy density and Lewis basicity to heavily fluorinated compounds, making dissolution of metal ions and organic molecules challenging. Functionalization is further complicated by colloidal instability toward heat and pH, as well as limited availability of biocompatible surfactants.We have devised new fluorous chelators that overcome solubility barriers and are able to bind a range of metal ions with high thermodynamic stability and biocompatibility. NE harboring chelators in the fluorous phase are a powerful platform for the development of multimodal imaging agents. These compositions rapidly capture metal ions added to the aqueous phase, thereby functionalizing NEs in useful ways. For example, Fe 3+ encapsulation imparts a strong paramagnetic relaxation effect on 19 F T 1 that dramatically accelerates 19 F MRI data acquisition times and hence sensitivity in cell tracking applications. Alternatively, 89 Zr encapsulation creates a sensitive and versatile PET probe for inflammatory macrophage detection. Adding lanthanides, such as Eu 3+ , renders NE luminescent. Beyond chelators, this Account further covers our progress in formulating NEs with fluorophores, such as cyanine or BODIPY dyes, with their utility demonstrated in fluorescence imaging, biosensing, flow cytometry and histology. Fluorous dyes soluble in FC oils are also key enablers for nascent whole-body imaging technologies such as cryo-fluorescence tomography (CFT). Additionally, fluorous cell-penetrating peptides inserted on the NE surface increase the uptake of NE by ∼8fold in weakly phagocytic stem cells and lymphocytes used in immunotherapy, resulting in significant leaps in detection sensitivity in vivo.
Fluorine-19 magnetic resonance imaging (MRI) has gained considerable momentum as a promising imaging modality for in vivo tracking of cellular therapies and as a diagnostic for inflammatory disease. To further the utility of this technique, we increase imaging probe sensitivity by merging paramagnetic metal chelates with aqueous perfluorocarbon (PFC) nanoemulsions. We prepared a highly fluorinated ferric tris(β-diketonate) chelate (MW = 1265.2 g/mol) at gram scale. This iron chelate is soluble in multiple PFC oils used for MRI and readily reduces the 19 F longitudinal relaxation time (T 1 ) to <100 ms with modest line broadening and displays superior properties for 19 F MRI applications. The sensitivity enhancement by Fe(III) laden PFC nanoemulsion was confirmed in MRI phantom studies, where reduced T 1 speeds data acquisition thereby increasing the 19 F image sensitivity per time via signal averaging. Additionally, 19 F relaxivity of nanoemulsions incorporating other metal ions, including Gd, Er, Ho, Dy, Mn, Cr, and Ni, were evaluated. Highmoment lanthanide ions, such as Gd(III), display severe line broadening, but other ions [e.g., Ho(III)] induce pseudocontact chemical shifts (up to 0.5 ppm) of 19 F in nanoemulsion, which makes them potentially useful for multichromatic 19 F imaging. Formulated nanoemulsions have a shelf life >200 days. Free β-diketonate or its iron complex in formed PFC nanoemulsion did not induce cytotoxicity in intracellularly labeled macrophages. Overall, ferric tris(β-diketonate) chelate provides a scalable approach for boosting sensitivity of PFC-based 19 F MRI probes. More generally, it can functionalize PFC oil, whose chemical modification remains challenging.
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