A Hyperfluorinated Hydrophilic Molecule for Aqueous 19F MRI Contrast Media

Fluorine-19 (19F) magnetic resonance imaging (MRI) has the potential for a wide range of in vivo applications but is limited by lack of flexibility in exogenous probe formulation. Most 19F MRI probes are composed of perfluorocarbons (PFCs) or perfluoropolyethers (PFPEs) with intrinsic properties which limit formulation options. Hydrophilic organofluorine molecules can provide more flexibility in formulation options. We report herein a hyperfluorinated hydrophilic organoflourine, ET1084, with ∼24 wt. % 19F content. It dissolves in water and aqueous buffers to give solutions with ≥8 M 19F. 19F MRI phantom studies at 9.4T employing a 10-minute multislice multiecho (MSME) scan sequence show a linear increase in signal-to-noise ratio (SNR) with increasing concentrations of the molecule and a detection limit of 5 mM. Preliminary cytotoxicity and genotoxicity assessments suggest it is safe at concentrations of up to 20 mM.


Introduction
MRI is currently the most powerful technique devoid of ionizing radiation for noninvasive clinical interrogation of the state of disease in soft tissue. Following the report of the first proton ( 1 H) MRI in 1973 [1], the technique quickly underwent several technological advances. Today, high resolution 3D anatomical images of all soft tissue types [2] can be obtained routinely in clinics across the globe. Obtaining medical information at the cellular and molecular levels by 1 H MRI often requires the use of contrast agents, and a variety of these are currently in use [3]. 1 H MRI contrast agents (CAs) generate contrast in vivo by altering the relaxivity of 1 H spins in surrounding water molecules but suffer from low SNR due to high background signal from water in soft tissue [4]. e first 19 F MRI images were reported in 1977 [5], but the platform received little attention as a clinical imaging technique until 2005 when Ahrens et al. demonstrated its potential for in vivo cell tracking [6]. Since then, several exogenous PFC and PFPE probes have been used successfully to track different cell types in vivo by 19 F MRI. ese include dendritic cells (DCs) in humans [7], T cell studies to track inflammatory events in a rodent model of type 1 diabetes [8], endogenous monocytes and macrophages in inflammatory lesions [9], macrophage distribution and density in mammary tumors and lung metastases [10], as well as lung imaging [11]. Other applications such as molecular imaging of thrombus and angiogenesis [12] have also been assessed. 19 F MRI contrast agents are superior to 1 H MRI because there is no endogenous MR detectable 19 F in soft tissue.
ere is therefore negligible tissue background signal, resulting in images with superior SNR. Recent advances in 19 F MRI technology including improvements in radiofrequency (RF) coil design, the development of dual 19 F/ 1 H imaging, as well as more advanced scan protocols [13] have greatly reduced scan times and improved image processing. Instruments with the ability to simultaneously capture complementary high resolution anatomical 1 H MR images alongside 19 F MRI hot-spots in one imaging session are also currently available [14]. However, the advances in instrumentation, scan protocols, and image analysis have not been matched by similar developments in biocompatible exogenous 19 F probes with flexible/diverse in vivo applications [15]. Apart from PFCs and PFPEs, several other fluorinated CAs including dendrimers, fluorinated amphiphiles, and hyperfluorinated molecules (such as PER-FECTA) have been reported. ese were examined in a recent review by Tirotta et al. [16].
To date, PFCs and PFPEs constitute the most common ingredient in exogenous 19 F MRI probes. However, their high hydrophobicity limits formulation flexibility and wide in vivo applicability. More recent research efforts in fluorinated CAs are gradually shifting towards hydrophilic molecules due to perceived flexibility in formulation and applicability. Several fluorinated hydrophilic polymers containing up to 20 % fluorinated monomer units while maintaining water solubility have been reported [17][18][19][20]. However, these commonly have a 19 [21]. In a recent report, we demonstrated that small hydrophilic nonionic organofluorine molecules have the potential for facile formulation into a myriad of 19 F MRI probes with unique 19 F MR signatures [22]. We present herein the synthesis and characterization of a new hyperfluorinated nonionic organofluorine molecule, ET1084, with 19 F content of ∼24 wt. %. is compound dissolves in water and aqueous buffers, yielding solutions of ≥8 M 19 F atoms which generate 19 F MR images with no chemical shift artifacts. Preliminary evaluation of cytotoxicity in RAW264.7 (a monocyte cell line) and ImKC (a Kupffer cell line) cells and genotoxicity in E. coli suggests that it is nontoxic at concentrations of up to 20 mM.

Chemical Synthesis
Procedures similar to our previously reported [23] general chemical synthesis procedures were employed. 2,2,3,3-Tetrafluoro-1,4-butanediol was purchased from Exfluor Research Corp., Round Rock, TX, USA. All other reagents were obtained from Sigma-Aldrich and used without further purification. Proton nuclear magnetic resonance ( 1 H NMR) spectra were recorded at 600 MHz on a Bruker 600 NMR spectrometer or at 300 MHz on a Bruker 300 NMR spectrometer. Carbon nuclear magnetic resonance ( 13 C NMR) spectra were recorded at 151 MHz on a Bruker 600 NMR spectrometer or at 75 MHz on a Bruker 300 NMR spectrometer. Fluorine nuclear magnetic resonance ( 19 F NMR) spectra were recorded at 282 MHz on a Bruker 300 NMR spectrometer. Chemical shifts are reported in parts per million (ppm) from an internal standard of acetone (2.05 ppm), chloroform (7.26 ppm), or water (4.79 ppm) for 1 H NMR and from an internal standard of either residual acetone (206.26 ppm), chloroform (77.00 ppm), or dimethyl sulfoxide (39.52 ppm) for 13 C NMR. NMR peak multiplicities are denoted as follows: s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), bs (broad singlet), dd (doublet of doublet), tt (triplet of triplet), ddd (doublet of doublet of doublet), and m (multiplet). Coupling constants (J) are given in hertz (Hz). High resolution mass spectra (HRMS) were obtained from the Mass Spectrometry Unit of the Bioscience Research Collaborative at Rice University, Houston, Texas. in layer chromatography (TLC) was performed on silica gel 60 F254 plates from EMD Chemical Inc., and components were visualized by ultraviolet light (254 nm) and/or phosphomolybdic acid, 20 wt.% solution in ethanol. SiliFlash silica gel (230-400 mesh) was used for all column chromatography.

Compound 1: Bis
To a mixture of xylitol (50.0 g, 329 mmol), dimethoxy acetone (60.0 mL), and methanol (100 mL) in acetone (2000 mL), p-toluenesulfonic acid monohydrate (5.65 g, 32.8 mmol) was added followed by vigorous stirring. All solids were dissolved after vigorous stirring for approximately 3 h, and the reaction mixture was stirred at room temperature overnight. K 2 CO 3 (4.54 g, 32.8 mmol) was added and stirred for 30 mins. Undissolved solids were filtered off, and the ensuing filtrate was concentrated in vacuo. e resultant colorless oil was chromatographed on silica gel eluted with 40% ethyl acetate/pentane to yield secondary alcohol 1 (64.8 g, 279 mmol, 85%) as a colorless oil. 1

Compound 2: Bis
To a solution of the alcohol 1 (50.0 g, 215 mmol) in CH 2 Cl 2 (1000 mL) under N 2 atmosphere, trimethylamine (90.0 mL, 646 mmol) was added and the resultant solution was cooled to 0°C for 15 mins. Methanesulfonyl chloride (21.7 mL, 280 mmol) was added, and the mixture was warmed to ambient temperature with stirring for 1 h after which the reaction was shown to be complete by TLC. e mixture was poured into saturated NH 4 Cl solution (1000 mL) with a separatory funnel. e organic phase was separated, and the aqueous phase was reextracted with CH 2 Cl 2 . e combined organic extracts were rinsed with brine, dried over Na 2 SO 4 , and concentrated by rotary evaporation to obtain a brown paste which precipitated upon addition of diethyl ether. e precipitate was filtered to give mesylate 2 (41.9 g, 176 mmol, 82%) as a pale yellow crystalline solid. 1

Cell
Culture. RAW264.7 cells were cultured in DMEM containing 4.5 g/L glucose, L-glutamine, and sodium pyruvate and supplemented with 10% heat-activated FBS and 1% P/S. ImKCs were cultured in a RMPI-1640 medium containing L-glutamine and supplemented with 10% heatinactivated FBS and 1% P/S. E. coli was cultured in a growth medium supplied with the SOS-Chromotest kit. All cells were incubated in a humidified atmosphere with 5% CO 2 at 37°C.

Cytotoxicity: MTS/PMS Assay for Determination of LC50 and Cell
Viability. RAW264.7 or ImKCs (0.01 × 10 6 cells/well) were plated in then treated at different concentrations of ET1084, BMH-21 (positive control), or left untreated (negative control) for 24 h. Dehydrogenase activity in the cultured cells was assayed using CellTiter 96R Aqueous Non-Radioactive Cell Proliferation assay kit obtained from Promega (Madison, WI, USA), according to the manufacturer's protocol. Briefly, cells were washed with PBS and then dosed with combined MTS/PMS solution in a fresh culture medium. Following incubation of the dosed cells for 4 hours at 37°C, the absorbance was measured immediately at λ � 450 nm using a multimode microplate reader (Filter Max F5, Molecular Devices).

Genotoxicity.
e genotoxic potential of ET1084 was determined using the SOS-Chromotest version 6.5 obtained from EBPI (Mississauga, Ontario, Canada) with modification. Briefly, E. coli was hydrated in a growth medium and incubated for 15 h in a humidified atmosphere with 5% CO 2 at 37°C. e absorbance (at 600 nm) of the turbid overnight bacteria suspension was adjusted to 0.055 using a fresh growth medium. e bacterial suspension (0.055 OD, 100 mL) was homogenized with or without ET1084 at final concentrations of 0, 10, 15, 20, and 30 mM or 4-NQO (positive control) content in a 96welled plate. Following 2 h incubation under conditions mentioned earlier, blue chromogen substrate (p-nitrophenyl phosphate in blue chromogen solution, 100 µL) solution was added to the inoculated medium and further incubated for 1.5 h. e experiment was quenched by addition of stop solution (50 µL), and genotoxicity (β-galactosidase activity) was measured at 595 nm; meanwhile, viability (alkaline phosphatase activity) was measured at 405 nm using a multimode microplate reader (Filter Max F5, Molecular Devices). e induction factor (IF) which is a correlation of the β-galactosidase and alkaline phosphatase activities was used to define the degree of genotoxicity [24,25].

Statistical Analysis.
Experiments were performed in triplicate, and the mean was calculated with standard deviation. Dose-response data for LC50 determination were analyzed by the probit method using Finney's table [26].
e LC50 values for RAW264.7 and ImKCs were determined by applying regression equation analysis to the probit-transformed data of mortality using Excel spreadsheet [27].

Results and Discussion
ET1084 was designed to have a tertiary ring conformation with a hydrophilic surface composed of highly soluble and biocompatible xylitol over a hydrophobic core bearing 19 F atoms with close-to-identical magnetic resonance frequencies.
e close-to-identical magnetic resonances are a requisite to 19 F MR images devoid of chemical shift artifacts [14]. As shown in Scheme 1, the compound was readily accessed from xylitol in four high-yielding synthetic steps. First, the terminal vicinal alcohols of xylitol were capped with acetone using standard acetonide protection conditions to obtain alcohol 1. e hydroxyl group of 1 was activated to obtain mesylate 2, which was heated in the presence of excess preformed 2,2,3,3-tetrafluoro-1,4butanedialkoxide to obtain compound 3. e alkoxide of compound 3 reacted with Hexakis(bromomethylbenzene) to obtain compound 4, which was deprotected under acid conditions to obtain ET1084.
e structures of all intermediates and the final product were confirmed by 1 H NMR, 19 F NMR (where applicable), and 13 C NMR as well as HRMS.
ET1084 dissolves readily in water and aqueous buffers including phosphate-buffered saline (PBS), histidine/saline, and acetate buffers to give clear solutions of ≥8 M 19 F at room temperature. e 19 F NMR spectrum (Figure 1(a)) shows two peaks at −121.4 ppm (separated by 0.3 ppm units). A single pulse frequency sweep from a 9.4T small animal imaging instrument shows a single peak which leads to 19 F MRI images with no chemical shift artifacts. Phantom dilution studies using 1Tand 9.4T instruments (see Supporting Materials S2) suggested that a concentration range between 0 and 200 mM was optimal for the characterization of the molecule at 9.4T. 19 F MRI scans performed on solutions of the molecule at concentrations of 1, 5, 10, 25, 50, 100, and 200 mM (Figure 1(b)), employing a 10-minute multislice multiecho (MSME) scan protocol (excitation bandwidth � 2000 Hz, TR � 2000 ms, and TE � 8.95 ms), showed a clear signal at concentrations as low as 5 mM with an observed SNR of 8.4 at this concentration. A plot of SNR against concentration (Figure 1(c)) showed a linear relationship (r 2 � 0.99) at concentrations up to 200 mM. Beyond this concentration (see Supporting Materials S2), the SNR starts dropping. Analyses of the spin-lattice relaxation time (T 1 ) and the spin-spin relaxation time (T 2 ) at different concentrations of the molecule (Figure 1(d)) suggest a T 1 of ∼450 ms and T 2 s, which show significant shortening with increasing concentration. is drop in SNR at concentrations >200 mM is rationalized by the spin-echo pulse sequence employed in acquiring the images. In 19 F MRI, using a spin-echo scan protocol, the signal intensity (I) can be related to the number of 19 F nuclei (N), the relaxation times (T 1 and T 2 ), and the scan parameters TR (repetition time) and TE (echo time) by [28] I � N(F) 1 − 2e −(TR−TE/2)/(2T 1 ) + e −TR/T 1 e −TE/T 2 . (1) It should be noted that in an aqueous medium where the concentration of water is ∼55 M compared to millimolar concentrations of the molecule, the spin-lattice relaxation time, T 1 , will remain fairly constant within the millimolar range for the 19 F nuclei. However, the spinspin relaxation time, T 2 , determined by the strength of dipolar interaction between neighboring 19 F nuclei and proton nuclei, experiences more significant changes with every increase in concentration of 19 F species. An increase in dipolar interactions between 19 F nuclei with increasing concentration causes faster relaxation of the nuclei. is in turn translates to the observed shortening in T 2 s. Based on Equation (1), this should lead to a decrease in signal intensity. However, the signal intensity is also directly proportional to the number of 19 F nuclei, so an increase in concentration should result in a corresponding increase in signal intensity. e overall effect on the signal intensity is therefore a trade-off between T 2 and effective concentration of 19 F nuclei. e observed increase in SNR with increasing concentration up to 200 mM and drop thereof suggest that the effect on the signal intensity is dominated by the effective concentration of 19 F nuclei within this range and the T 2 effect dominates thereafter.
For in vivo applications, high concentrations of ET1084 are required at the target to allow conspicuity and this is achievable by nanoparticle formulation. Its high aqueous solubility makes liposome formulation an attractive option. Liposomes are cleared through the monocyte-phagocyte system (MPS). As a result, leucocytes and resident immune cells in the liver and spleen are more likely to encounter high concentrations of the compound. We therefore chose a monocyte cell line and a Kupffer cell line to preliminarily assess the toxicity of the compound. RAW264.7 (monocyte cell line) and ImKCs (Kupffer cell line) were exposed to different concentrations of ET1084 over 24 hours. Regression graphs of probit mortality of both cell types plotted against log values of increasing concentrations of the compound (Figure 2(a)) suggest that the LC50 of the compound is 52.44 mM for the RAW264.7 cells and 31.68 mM for the ImKCs. In addition, the data suggest that the compound is innocuous to the RAW264.7 at concentrations up to 20 mM and cell viability of over 80 % was observed for the ImKCs at the same concentration (Figure 2(c)). e potential of ET1084 to react with genetic material was also assessed using the SOS-Chromotest kit, which allows for the detection of DNA-damaging agents in Escherichia coli (E. coli). Figure 2(

Conclusion
In summary, we have synthesized and characterized a novel hydrophilic hyperfluorinated organofluorine molecule amenable to aqueous formulations of exogenous probes for in vivo 19 19 F NMR spectrum from a 300 MHz instrument shows two peaks separated by 0.3 ppm and a single peak from a single pulse frequency sweep in a 9.4T small animal imaging instrument which generates a sharp 19 F MRI image devoid of chemical shift artifacts; (b) dilution studies show that the compound is visible at concentrations of 5 mM from a 10-minute MSME scan at 9.4T, (c) plot of SNR against concentration shows linearity (R 2 � 0.99); (d) T 1 and T 2 relaxation times estimated by 19 F MRI at 9.4T (details on scan parameters in SI). Data reported as mean ± SD. of 150 nm. e preparation of both targeted and nontargeted liposome formulations of this compound for in vivo assessment is ongoing and will be reported in the future.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.