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

Abstract

Increased emphasis on personalized medicine and novel therapies requires the development of noninvasive 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 noninvasive and has no associated radioactivity, yet penetrates deep tissue. We now demonstrate a novel class of dual (1)H/(19)F 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 (19)F NMR signal and exposure to β-galactosidase induces a large (19)F NMR chemical shift response. In the presence of ferric ions, the liberated aglycone generates intense proton MRI T(2) contrast. The dual modality approach allows both the detection of substrate and the imaging of product enhancing the confidence in enzyme detection.

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.

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.

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).

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).

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.

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.