Dual ¹⁹F/¹H MR gene reporter molecules for in vivo detection of β-galactosidase

Abstract

Increased emphasis on personalized medicine and novel therapies require the development of non-invasive strategies for assessing biochemistry in vivo. The detection of enzyme activity and gene expression in vivo is potentially important for the characterization of diseases and gene therapy. Magnetic resonance imaging (MRI) is a particularly promising tool since it is non-invasive, and has no associated radioactivity, yet penetrates deep tissue. We now demonstrate a novel class of dual ¹H/¹⁹F nuclear magnetic resonance (NMR) lacZ gene reporter molecule to specifically reveal enzyme activity in human tumor xenografts growing in mice. We report the design, synthesis, and characterization of six novel molecules and evaluation of the most effective reporter in mice in vivo. Substrates show a single ¹⁹F NMR signal and exposure to β-galactosidase induces a large ¹⁹F NMR chemical shift response. In the presence of ferric ions the liberated aglycone generates intense proton MRI T₂ contrast. The dual modality approach allows both the detection of substrate and imaging of product enhancing the confidence in enzyme detection.

INTRODUCTION

Several reporter proteins have been used in gene expression and regulation studies including β-galactosidase (β-gal), firefly luciferase (luc), β-glucuronidase, fluorescent proteins (such as green fluorescent protein, GFP), transferrin and ferritin, the enzymes creatine and arginine kinase, tyrosinase and polycations such as poly lysine ^{1, 2}. Historically, the lacZ gene encoding β-gal has been widely used with applications ranging from molecular biology to small animal investigations and clinical trials including assays of clonal insertion, transcriptional activation, protein expression, and protein interaction ^{3, 4}. Recently, various innovative approaches to assessing β-gal activity in vivo have been presented exploiting gadolinium contrast enhanced ¹H magnetic resonance imaging (MRI) or ¹⁹F NMR ^5-12, optical ^13-18 and radionuclide imaging ^19-21. Traditional ¹⁹F NMR spectroscopy approaches have the advantage of detecting both the substrate and product simultaneously ^7-9, but while imaging is feasible ^{22, 23}, the achievable signal to noise has so far been insufficient for imaging in animals. By comparison ¹H MRI contrast can be far more sensitive, but presence of substrate and formation of product may not be readily identifiable due to tissue heterogeneity. We have demonstrated an Fe(III)-based ¹H MRI approach using the commercially available black histological stain sodium 3,4-cyclohexenoesculetin-β-D-galactopyranoside (S-Gal™ sodium salt) to detect β-gal activity in stably transfected lacZ expressing cancer cells in vitro and in vivo and this approach was also used to label and track stem cell localization ^{24, 25}. It occurred to us that the approaches could be combined, whereby ¹⁹F NMR spectroscopy would define substrate accumulation and conversion, while iron based ¹H MRI contrast reveals regional enzyme activity.

Escherichia coli (lacZ) β-gal catalyses the hydrolysis of β-D-galactopyranosides by cleavage of the C-O bond with β-configuration between D-galactose and the aglycone. Considering the multiple requirements for an enzyme responsive ¹⁹F/¹H MRI indicator, we chose salicylaldehyde nicotinoyl hydrazone, salicylaldehyde isonicotinoyl hydrazone and salicylaldehyde benzoyl hydrazone as aroylhydrazone chelators based on reported characteristics ^26-28. A fluorine atom introduced at the ortho or para position to the C_1(gal)-O bond in the salicylaldehyde fragment was expected to display a large ¹⁹F NMR chemical shift in response to cleavage by β-gal ²⁹.

We now report proof of principle using a fluorosalicylaldehyde aroylhydrazone β-D-galactopyranoside, which provides a 19F NMR signal sensitive to β-gal enzyme cleavage and the liberated aglycone spontaneously traps ferric ions generating ¹H MRI contrast on T₂W images (Fig. 1). We currently add ferric ammonium citrate, but contrast could in principle develop from entrapment of endogenous Fe³⁺.

Figure 1. Proposed mode of action.

β-galactosidase activity is detected by ¹⁹F NMR chemical shift accompanying release of aglycone and ¹H MRI contrast induced by ferric ion complex.

EXPERIMENTAL PROCEDURES

Detailed molecular characterization results based on chemical shift assignment, and chemical analyses are provided in supporting materials. All in vivo studies were performed with approval from the Institutional Animal Care and Use Committee.

The synthetic route and structures of 1-17 are shown in Fig. 2. Reaction of 2, 3, 4, 6-tetra-O-acetyl-α-D-galactopyranosyl bromide 1 (Sigma Chemical Company, St Louis, MO) with 3- or 5-fluorosalicylaldehydes 2 or 3 at 50 °C catalyzed by tetrabutylammonium bromide (TBAB) in an aqueous dichloromethane biphasic system (pH 8~9) under N₂ afforded 2-[(2′, 3′, 4′, 6′-tetra-O-acetyl-β-D-galactopyranosyl)oxy]-3 or 5-fluorobenzaldehydes 4 or 5 in high yields (82~92%). Treatment of 4 or 5 respectively with 1.1 equivalents of benzoic hydrazide, nicotinic hydrazide or isoniazid in acidic medium at 80 °C produced the corresponding 2-[(2′, 3′, 4′, 6′-tetra-O-acetyl-β-D-galactopyranosyl)oxy]-3 or 5-fluorobenzaldehyde aroylhydrazones 6~11 in 90~100% yields, which were deacetylated with NH₃/MeOH from 0 °C to room temperature giving their free galactopyranosides 12~17 in quantitative yields.

Figure 2. The reactions and the structures of 1~17.

Reaction conditions: (a) CH₂Cl₂-H₂O, pH 8~9, 50 °C, TBAB, N₂, 5~6 hr, 92%(→4) or 86%(→5), respectively; (b) EtOH, AcOH (20 μL), benzoic hydrazide, nicotinic hydrazide or isoniazide (1.1 equiv.), 80°C, N₂, 4~5 hr, 100%(→6), 90%(→7), 95%(→8), 95%(→9), 100%(→10) and 93%(→11) respectively; (c) 0.5M NH₃-MeOH, 0°C→r.t., 24 hr, quantitative yields.

Detailed syntheses

2-[(2′, 3′, 4′, 6′-Tetra-O-acetyl-β-D-galactopyranosyl)oxy]- 3 or 5- fluoro-benzaldehydes 4~5

General Procedure. A solution of 2, 3, 4, 6-tetra-O-acetyl-α-D-galactopyranosyl bromide 1 (615 mg, 1.5 mmol) in CH₂Cl₂ (10 mL) was added dropwise to a vigorously stirred biphasic mixture (pH 8~9) of 3- or 5-fluorosalicylaldehyde (2~3) (252 mg, 1.8 mmol) and tetrabutylammonium bromide (TBAB) (160 mg, 0.5 mmol) in CH₂Cl₂-H₂O (20 mL, 1:1 V/V’) over a period of 1 hr at 50 °C under N₂, and the stirring continued for 4~5 hr until TLC showed complete reaction. The products were extracted with CH₂Cl₂ (4×25 mL), washed, dried (Na₂SO₄), evaporated under reduced pressure to give a syrup, which was purified by column chromatography on silica gel to give 2-[(2′, 3′, 4′, 6′-tetra-O-acetyl-β-D-galactopyranosyl)oxy]-3 or 5-fluorobenzaldehydes 4~5, as white crystals in >85% yield.

2-[(2′, 3′, 4′, 6′-Tetra-O-acetyl-β-D-galactopyranosyl)oxy]-3 or 5-fluorobenzaldehyde aroylhydrazones 6~11

General Procedure. A solution of 2-[(2′, 3′, 4′, 6′-tetra-O-acetyl-β-D-galactopyranosyl)oxy]-3 or 5-fluorobenzaldehyde 4 or 5 (200 mg, 0.44 mmol) in anhydrous EtOH (15 mL) containing AcOH (20 μL) was stirred vigorously respectively with benzoic hydrazide, nicotinic hydrazide or isoniazid (0.48 mmol, 1.1 equiv.) at 80 °C under N₂ until TLC showed that the reaction was complete, then coevaporated with toluene to dryness in vacuo. Crystallization from EtOH-H₂O or chromatography of the crude syrup on silica gel with appropriate eluents yielded the corresponding 2-[(2′, 3′, 4′, 6′-tetra-O-acetyl-β-D-galactopyranosyl)oxy]-3 or 5-fluorobenzaldehyde aroylhydrazones 6~11 in 90~100% yields.

2-[(β-D-galactopyranosyl)oxy]-3 or 5-fluorobenzaldehyde aroylhydrazones 12~17

General Procedure. A solution of 2-[(2, 3, 4, 6-tetra-O-acetyl-β-D-galactopyranosyl)oxy]-3 or 5-fluorobenzaldehyde aroylhydrazones 6~11 (200 mg) in anhydrous MeOH (20 mL) containing 0.5M NH₃ was vigorously stirred from 0 °C to room temperature overnight until TLC showed complete reaction, then evaporated to dryness in vacuo. Chromatography of the crude syrup on silica gel with EtOAc-MeOH afforded the corresponding free β-D-galactopyranosides 12~17 in quantitative yields.

3-fluorosalicylaldehyde nicotinoyl hydrazone (3-FBNH)- hydrolysis product of 13

A solution of 3-fluorosalicylaldehyde (330 mg, 2.36 mmol) in anhydrous EtOH (15 mL) containing AcOH (20 μL) was stirred vigorously with nicotinic hydrazide (323 mg, 2.36 mmol) at 80 °C under N₂ until TLC showed that the reaction was complete, then coevaporated with toluene to dryness in vacuo. Crystallization from EtOH-H₂O yielded 3-FBNH (526 mg) as white needles.

Fe:3-FBNH Complex. To a solution of 3-FBNH (500 mg, 1.93 mmol) and Et₃N (195 mg, 1.93 mmol) in anhydrous MeOH (100 mL) was added dropwise a solution of Fe(ClO₄)₃·6H₂O (446 mg, 0.97 mmol) in anhydrous MeOH (50 mL) with stirring and gentle reflux under N₂ for 30 min. Upon cooling, a fine black precipitate was obtained, which was filtered, washed with EtOH then Et₂O, and dried in vacuo yielding Fe-3-FBNH Complex [Fe(3-FBNH-H)₂](ClO₄)·3H₂O (576 mg) as black powder. Anal. Calcd. for C₂₆H₂₄ClFeN₆O₁₁F₂ (%): C, 43.03, H, 3.34, N, 11.59; Found: C, 42.95, H, 3.41, N, 11.48.

Detection of β-galactosidase by ¹⁹F NMR in solution 2-[(β-D-galactopyranosyl)oxy]-3-fluorobenzaldehyde nicotinoyl hydrazone 13, 2-[(β-D-galactopyranosyl)oxy]-3-fluorobenzaldehyde isonicotinoyl hydrazone 14 (2.11 mg, 5.0 μmol) or 2-[(β-D-galactopyranosyl)oxy]-5-fluorobenzaldehyde benzoylhydrazone 15 (2.10 mg, 5.0 μmol) were dissolved in PBS (595 μL) and a solution of β-gal (5 μL, 1 unit/μL; E801A, Aldrich) was added. ¹⁹F NMR time course spectra were acquired immediately at 376 MHz in 102 s each and continued at 37 °C over 4 hrs to assess relative rates of activity. 13 was also examined by ¹⁹F MRI. In this case 13 (7 mg) was dissolved in PBS/DMSO (250 μL 1:1 mixture) at 37 °C and β-galactosidase (E801A 20 units) added. ¹⁹F MR images were acquired at 376 MHz with 930 μm in plane resolution across a 30 mm × 30 mm, field of view and 10 mm slice thickness in 4 minutes 16 seconds each using 8 averages at each phase encode and TR =1000 ms, TE=1.6 ms and 90 deg flip angle.

¹H MRI T₂ maps of phantoms of 13 and S-Gal in agar

Mixtures of agar and 3,4-cyclohexenoesculetin-β-D-galactopyranoside sodium (S-Gal™ sodium salt, Sigma-Aldrich Inc., St. Louis, MO) (5 mM, 40 μl) + ferric ammonium citrate (FAC 2.5 mM, 40 μl) or 13 (5 mM, 40 μl) + FAC (2.5 mM, 40 μl) with or without β-gal (E801A, 5 units) were prepared in sections of 384 well plates cut to fit in a 1 turn, 2 cm volume solenoid coil. T₂ maps were acquired at 4.7 T using a spin echo sequence with variable echo times. MRI parameters were: FOV= 40 mm × 40 mm, matrix 128×128, slice thickness= 1 mm, TR= 6 s, TE= 10, 20, 30, 50, 80, 100, 150, 200 ms.

Detection of β-galactosidase by ¹⁹F NMR in cells

Human MCF7 breast and PC3 prostate cancer cells were transfected to stably express the E.coli lacZ gene constitutively, as described previously ^{8, 9}. Wild type and -lacZ cells were grown in culture dishes under standard conditions and harvested for NMR tests. ¹⁹F NMR spectra were acquired at 9.4 T up to 40 hrs after addition of 13 (2.11 mg, 5.0 μmol) to MCF7 cells (5.0×10⁶) in 600 μl PBS and in some cases Fe³⁺ (FAC 2.5 μmol) was added. In addition to evaluating intact cells, additional tests were conducted with lysed cells. For comparison equal numbers of cells were prepared for evaluation intact or following lysis. Cell lysis was achieved by a freeze/thaw method: equal numbers of MCF7-lacZ or PC3-lacZ cells were suspended in PBS and then frozen at −80 °C for 10 mins before thawing at room temperature over 3 cycles.

In vivo MR studies

PC3 cells (wild type or transfected to stably express lacZ ⁸) were implanted subcutaneously in thighs of SCID mice (n=3). NMR studies were performed at 4.7 T using a Varian Unity INOVA horizontal bore scanner (200.1 MHz for ¹H, 188.2 MHz ¹⁹F). When the tumors reached ~0.8 cm in diameter, mice were anesthetized (isoflurane/air) and placed on a platform with the tumor bearing leg inserted into a 2 cm diameter home built volume coil (tunable from 200.1 MHz for ¹H to 188.2 MHz for ¹⁹F). The animal temperature was maintained at 37 °C by a warm pad with circulating water. The mouse bed was inserted into the bore of the MR scanner and shimming was performed on the tissue water proton signal. T₁ and T₂ maps of the tumor were measured using a spin echo sequence with varying repetition and echo times (acquisition parameters: field of view (FOV) 50 mm × 50 mm, matrix 128×128, slice thickness 1 mm, 15 slices). The mouse-bearing platform was removed from the magnet and a solution of 13 (50 μL 50 mM, DMSO/PBS 1:1 V/V’) was injected directly into the tumor in a “fan” pattern. The platform was replaced in the magnet and ¹⁹F NMR spectra were obtained immediately after retuning the coil to the ¹⁹F resonance frequency. Each spectrum was acquired in 166 s (acquisition parameters: pulse width 45 μs, 128 acquisitions, spectral width 100 ppm, 60 Hz exponential line broadening). The mouse-bearing platform was again removed from the magnet and a solution of FAC (50 μL 50 mM, PBS) was now injected directly into the tumor in a similar pattern to 13. ¹⁹F NMR spectra were obtained and the coil was retuned to the ¹H frequency and new T₁ and T₂ maps were acquired. For analysis, regions with injected contrast agent were identified based on the T₂- and T₁-weighted images (to delineate tumor boundary and locate the injected Fe³⁺ ions, respectively). For one animal, no injection site could be identified inside the tumor and hence the data for this tumor were not used. To confirm β-gal activity in tumors, tissues were embedded in Tissue-Tek OCT (Miles Laboratory, Elkhart, IN, USA), and frozen in liquid nitrogen. Cryostat sections (8 μm) were collected on gelatin-coated glass slides and stained with X-gal and eosin (Sigma) for β-gal activity.

Testing product aglycone visibility in vivo

A mixture of sodium trifluoroacetate (50mM) and aglycone (3-FBNH (3 or 6 mg in total volume 50 μl or 100 μl of a 3:1 mixture of DMSO:PBS) was injected into muscle of three adult 129S-Gt(ROSA)26Sor/J mice (2 live and 2 post mortem). ¹⁹F NMR spectra were acquired immediately at 4.7 T and every 2 mins. NaTFA served as a chemical shift reference at 0 ppm.

RESULTS

Each of the target reporter molecules was achieved in high yield and structures were confirmed by NMR and chemical analyses (see supporting materials). The anomeric β-D-configuration of compounds 4~17 in the ⁴C₁ chair conformation was confirmed by the observed ¹H-NMR chemical shifts (δ_H 4.79~5.12 ppm) of the anomeric protons and the J_1,2 (J ~8 Hz) and J_2,3 (J ~10 Hz) coupling constants ²⁹. The anomeric carbon resonances appeared at δ_C-1′ 100.05~105.23 ppm in accord with the β-D-configuration. ¹⁹F NMR chemical shifts were measured with respect to sodium trifluoroacetate (NaTFA, δ= 0 ppm) in a capillary as an external standard. 12~17 each gave a single narrow ¹⁹F NMR signal between δ_F −44.0 and −55.0 ppm (Table 1) essentially invariant (Δδ_F≤0.05ppm) with pH in the range 3 to 12 and temperatures from 25 to 37 °C in 0.9% saline or rabbit whole blood.

Table 1.

¹⁹F NMR chemical shifts (ppm) of 12~17 and hydrolytic rates (μmol/min/unit) of 13~15 with β-gal.^*

No.	12	13	14	15	16	17
δF (substrate)	−54.84	−54.62	−54.60	−44.38	−45.02	−45.03
δF (aglycone)	---	−62.27	−62.03	−49.61	---	---
Δ δ F	---	7.65	7.43	5.23	---	---
ν (μmol/min/unit)	---	3.91	1.67	0.55	---	---

^*β-gal (E801A) at 37 °C in PBS (0.1M, pH=7.4).

When β-gal (E801A) was added to 12-17 in phosphate buffered saline (PBS) at 37 °C, only 13~15 were hydrolyzed releasing the 3- or 5-fluorobenzaldehyde aroylhydrazones appearing also as single narrow ¹⁹F signals shifted upfield between Δδ_F=5.23~7.65 ppm (δ_F −49.0~−63.0 ppm) (Figs. 3, S1, Table 1), which is comparable to shifts reported previously for ¹⁹F NMR gene reporter molecules ^{7-9, 29, 30}. The hydrolysis of 13~15 proceeded smoothly indicating that the liberated aglycones 3- or 5-fluorobenzaldehyde aroylhydrazones had no inhibitory effects on β-gal, and the shapes of the kinetic curves suggest straightforward first-order kinetics. The rate of reaction of 13 (ν_(E801A) = 3.91 μmol/min/unit) was comparable to that reported previously for 3-O-(β-D-galactopyranosyl)-6-fluoropyridoxol (GFPOL) ³⁰ and much faster than the other molecules here, so it was chosen for further studies. Enzyme induced hydrolysis was also observable by ¹⁹F MRI (Fig. 3). Titration of product 3-fluorobenzaldehyde nicotinoyl hydrazone (3-FBNH) showed a small chemical shift (~0.5 ppm) in the pH range 6.5 to 7.7 (Fig. S2).

Figure 3. Substrate response to β-galactosidase.

Graph shows the loss of each of the three agents 13 - 15 (5.0 μmol; □ 13; ● 14; Δ 15) when incubated with β-galactosidase (E801A, 5 units) in PBS (0.1 M, 600 μL) at 37 °C, as detected by ¹⁹F NMR spectroscopy revealing 13 reacts fastest. When β-galactosidase (20 units) was added to a solution of 13 (7 mg 16.5 μmol) in 250 μL PBS/DMSO 1:1 at 37 °C conversion was detectable by ¹⁹F NMR spectroscopy and imaging at 9.4 T (376 MHz): baseline at left and after 2.5 hrs at right showing product 3-fluorobenzaldehyde nicotinoyl hydrazone (3-FBNH).

Proton MRI of agar phantoms of 13 or commercial S-Gal^® (3,4-cyclohexenoesculetin β-D-galactopyranoside) with ferric ions showed very similar T₂ (152 ± 21 ms vs. 156 ± 25 ms; Fig. 4). Incorporation of β-gal in the agar yielded much reduced T₂ values and the difference was much greater for 13 than S-Gal (23 ±12 ms vs. 78 ± 11 ms: alternately, ΔR₂ 36.9 s⁻¹ vs. 6.4 s⁻¹). By analogy with S-Gal ²⁴ we attribute the relaxation enhancement to formation of complex between 3-FBNH and Fe³⁺. Chemical analysis indicated a 1:2 Fe³⁺:3-FBNH complex.

Figure 4. ¹H MRI T₂ maps of phantoms of 13 and S-Gal in agar.

Comparison of T₂ shortening due to addition of β-gal enzyme to commercial substrate S-Gal or 13. A) S-Gal (5 mM) + Fe³⁺ (2.5 mM); B) 13 (5 mM) + Fe³⁺ (2.5 mM); C) as (A) + β-gal (E801A, 5 units); D) as (B) + β-gal (E801A, 5 units).

When 13 was incubated with MCF7 human breast tumor cells for 5 hr in PBS at 37 °C under 5% CO₂ in air, no changes were observed in the ¹⁹F NMR spectrum. Addition of 13 to stably transfected MCF7-lacZ cells led to cleavage in a smooth monotonic manner with a rate of 0.25 μmol/min per million cells, and appearance of a new ¹⁹F signal of the released aglycone 3-fluorobenzaldehyde nicotinoyl hydrazone (3-FBNH) around −62.27 ppm (Fig. 5). When ferric ammonium citrate (FAC) was added after 28 hrs the product aglycone ¹⁹F signal immediately disappeared, which we attribute to a paramagnetic relaxation enhancement in the Fe³⁺: 3-FBNH complex.

Figure 5. Detection of β-gal activity in cultured cells.

376 MHz ¹⁹F NMR spectra of 13 (2.11 mg, 5.0 μmol) after addition to stably transfected MCF7-lacZ cells (5.0×10⁶). Fe³⁺ (2.5 μmol) was added after 28 hr. Each spectrum was acquired in 205 s, and enhanced with an exponential line broadening 40 Hz.

Given that conversion of substrate appeared much slower in cells than with enzyme in vitro, tests were conducted to compare intact and lysed cells. Incubation of 13 with equal numbers of intact or lysed MCF7-lacZ cells showed 25% conversion after overnight incubation (14 hrs), whereas lysed cells showed 75%. Similarly, PC3-lacZ cells showed only 15% conversion, while lysed PC3-lacZ cells showed 85%.

As an initial proof of principle, 13 and FAC were injected into wild type (WT) and lacZ-transfected PC3 human prostate tumor xenografts growing in mice (Fig. 6). Spin lattice relaxation time (T₁)-weighted images showed a local hyperintensity associated with the ferric ions and a corresponding drop in the T₁ values was observed. For groups of tumors there was no significant difference between mean baseline T₁ (2.4 ± 0.4 s; n= 2 WT vs. 2.4 ± 0.3 s; n=3 lacZ; p>0.8). Following injection of contrast agent (i.e., 13 plus FAC), T₁ decreased in WT tumors (T₁ = 1.5 ± 0.4, p< 0.2), but the change was significant in lacZ tumors (T₁= 1.1 ± 0.4; p<0.01). For the same regions of the tumors a large signal decrease was observed in spin-spin (T₂)-weighted ¹H images in PC3-lacZ tumors only at the injection site. T₂ was similar before injection of contrast agent (T₂ = 55 ± 5 ms (lacZ) vs. T₂ = 52 ± 16 ms (WT)). Following administration of contrast agent T₂ changed significantly in lacZ tumors (T₂ = 39 ± 5 ms; p<0.05), but not in WT tumors (T₂ = 53 ± 15 ms; p>0.9). ¹⁹F spectroscopy showed the presence of the substrate 13 in the WT tumors, which decreased slowly (Fig. 7), but no aglycone product. In corresponding PC3-lacZ tumors, no substrate or product signal was observed by ¹⁹F NMR. Enzyme activity was confirmed post mortem based on X-gal staining of slices of PC3-lacZ tumors, which turned intense blue, whereas wild type tumors showed no blue color (Fig. 6).

Figure 6. Imaging β-gal activity in vivo.

In vivo study showing lacZ gene-reporter activity detected by ¹H MRI in representative PC3 tumor xenografts in SCID mice after intra-tumoral injection (100 μl total volume) of ferric ammonium citrate (FAC) and 13. The presence of FAC + 13 is seen in T₁-weighted images (TR/TE = 300/12 ms) and T₁ maps (white arrows) in both wild type tumors (columns 1 and 2, row 2) and lacZ tumors (columns 1 and 2, row 4). Baseline tumor T₁ = 2.8± 0.1 s for lacZ and 2.6± 0.2 s for WT, versus 1.1± 0.6 s for lacZ and 1.8 ± 0.5 s for WT following injection of 13 + FAC. Significant change in T₂-weighted images (TR/TE = 6000/50 ms, column 3, row 4) and T₂ values (column 4, row 4) was seen only in the lacZ-transfected tumors post injection of FAC and 13 (pink arrows). Baseline T₂ = 56 ± 4 ms for lacZ and T₂ = 63 ± 7 ms for WT, became T₂ = 34 ± 10 ms for lacZ and T₂ = 63 ± 18 ms for WT. Green arrow indicates anomalous injection outside tumor, and this region was excluded from analysis. Histological sections at right (original magnification × 100) confirm intense β-gal expression in –lacZ tumor based on X-gal and H&E staining (blue, lower slide) with essentially no activity in WT tumor (pink, upper slide).

Figure 7. Detection of β-gal activity in vivo by ¹⁹F NMR.

Left: Following injection of 13 (1 mg) into a PC3-WT tumor ¹⁹F NMR spectroscopy showed substrate at −54.6 ppm with respect to sodium trifluoroacetate reference (lower spectrum). Following injection of 0.6 mg FAC the substrate remained visible (upper left). Spectra were acquired in about 2½ minutes and enhanced with 60 Hz exponential line broadening prior to Fourier transformation giving a SNR of 35.

Right: Similar injection into PC3-lacZ tumor showed minimal ¹⁹F NMR signal and there was minimal change after addition of FAC (upper).

Upper spectra A mixture of sodium trifluoroacetate and aglycone (3-FBNH; 3 mg) in total volume 50 μl was injected into muscle of a dead adult 129S-Gt(ROSA)26Sor/J mouse. ¹⁹F NMR spectra were acquired immediately and every 2 mins. NaTFA served as a chemical shift reference at 0 ppm and remained quite constant. Meanwhile aglycone was initially seen with SNR = 194 at −62.8 ppm, but signal decreased and was no longer observed after 6 mins.

Lack of detectable ¹⁹F NMR could have been caused by clearance of the product from lacZ tumors in vivo or potentially precipitation of the product by association with ferric ions, rendering it NMR invisible. To gain further insight into the lack of ¹⁹F signal, product aglycone was injected into muscle of ROSA26 mice, which express lacZ throughout their bodies. Internal NaTFA standard and aglycone were immediately detectable, but over a few minutes the aglycone signal at −61.5 ppm disappeared in both live and dead mice, while TFA remained visible in the dead animal (Fig. 7).

DISCUSSION

We have demonstrated a novel dual ¹⁹F/¹H MR lacZ gene detection approach by introducing a fluorine atom into iron-chelating aroylhydrazone aglycones of β-D-galactopyranosides. When activated by β-gal, the released fluoroaroylhydrazones display a distinct chemical shift with respect to the substrate and can be spontaneously trapped by Fe³⁺ at the site of enzyme activity forming highly relaxing complexes in situ. The complexes cause strong T₂ relaxation, providing ¹H MRI contrast to reveal lacZ expression.

We believe the T₁ contrast following injection is attributable to free ferric ions from FAC and this was observed in both WT and lacZ tumors (Fig. 6). However, β-gal activity was clearly revealed by the co-localized hypointensity in T₂-weighted images and corresponding significant shortening in T₂, whereas no T₂ contrast was observed for WT tumors.

The utility of a reporter molecule is predicated on a reasonable rate of activity. The conversion rates in vitro were very slow (Fig. 3). Aglycone was released much more rapidly by lysed MCF7-lacZ or PC3-lacZ cells indicating that cell permeation is a strong barrier to activity. However, contrast was observed almost immediately in lacZ tumors upon direct intratumoral injection. Such differential rates of activity have also been reported for ¹⁹F reporters ^{8, 9}. The ultimate goal is development of reporter molecules which may be administered systemically. At this stage the method requires direct intra tumoral injection, but we note that this limitation has also been encountered by most other reports regarding reporter molecules for β-gal using NMR, radionuclides or photoacoustic tomography ^{8, 9, 12, 16, 21, 24}. To date systemic administration has generally been restricted to optical imaging approaches ^{14, 18, 31} and a single report regarding a Gd-based contrast agent ⁶.

Ideally ¹⁹F NMR would show both substrate and product and this was observed in cells (Fig. 5). However, the liberated product signal was absent in the presence of ferric ions and in vivo. This could be attributed to clearance of the product from lacZ tumors and ROSA26 muscle in vivo, but the post mortem test in the ROSA26 mouse also indicated signal disappearance over a period of 6 minutes consistent with precipitation of the product (possibly in association with endogenous ferric ions), which renders it NMR invisible. ¹⁹F signal for TFA remained visible over this time. The ¹⁹F NMR is able to locate substrate, but not product and we are seeking alternate molecules, which can reveal both substrate and product.

In the present studies we added exogenous ferric ions, but we note that cancer cells often exhibit elevated iron levels and a number of Fe-chelators have been proposed for cancer therapy ³². Indeed, aroyl hydrazones are in clinical use as ferric ion scavenging agents ³³ suggesting a potential for clinical theranostic application. There is an extensive literature on aroyl hydrazones regarding thermodynamic characteristics of ferric ion complexes ^{26, 28} and we assume the ¹⁹F analogs presented here behave similarly. We were not able to obtain a crystal structure of the complex, but chemical analysis confirmed a 2:1 structure.

¹⁹F NMR is gaining popularity as a reporter for diverse enzyme reactions based on various strategies. The simplest is change in chemical shift as we and others have exploited extensively ^{8, 9, 34, 35}. More recently differential relaxation rates have been exploited based on paramagnetic relaxation enhancement (PRE) ^{10, 36} or differential restricted mobility ^{37, 38} to reveal enzyme activity based on changes in line broadening. In addition, several recent reports have presented bimodal reporter strategies revealing enzyme activity based on ¹⁹F NMR and fluorescence ^{38, 39}.

Ultimately, we hope to develop this approach for systemic delivery of reporter molecules, but direct injection into the tissues of interest already demonstrates selective detection of lacZ expression versus WT. The multimodality approach represents a new paradigm exploiting ¹⁹F NMR together with both T₁ and T₂ ¹H MRI contrast to reveal the presence or absence of enzyme activity. The observations are in accord with renewed excitement and innovations in ¹⁹F NMR notably for detecting enzyme activity ^{37, 39-42}.