Reagents used for synthesis were purchased from Aldrich and used without further purification. Column chromatography was performed on silica gel (SiliCycle Inc., 60 Å, 40–63 mm) slurry packed into glass columns. Synthetic amyloid-β peptide (1–40/42) was purchased from rPeptide (Bogart, GA, 30622). 1H and 13C NMR spectra were recorded at 500 MHz and 125 MHz respectively, and reported in ppm downfield from tetramethylsilane. Fluorescence studies were carried out using a F-4500 Fluorescence Spectrophotometer (Hitachi). Transgenic APP-PS1 mice and age-matched wild-type mice were purchased from Jackson Laboratory. All experimental procedures were approved by the Subcommittee on Research Animal Care at Massachusetts General Hospital. In vivo NIR imaging was performed using the IVIS®Spectrum animal imaging system (Caliper LifeSciences, Hopkinton, MA), and spectral unmixing was conducted using LivingImage® 3.2 software.
Synthesis of CRANAD-3
The synthesis of the immediate material (2,2-difluoro-1,3-dioxaboryl-pentadione) was performed using a modified procedure 7
. The immediate compound (0.15g, 0.1mmol) was dissolved in acetonitrile (3.0 ml), and acetic acid (0.2ml), tetrahydroisoquinoline (0.04mL, 0.3mmol), and 6-N,N′
-diethyl-3- pyridylaldehyde (0.36g, 2.0mmol) were added. The resulting solution was stirred at 60°C overnight. A black residue was obtained after removing the solvent, and was subjected to flash column chromatography with methylene chloride to produce a black powder (63.0mg, yield: 15.0%). 1
H and 13
C NMR spectra were recorded at 500 MHz and 125 MHz respectively, and reported in ppm downfield from tetramethylsilane.1
H NMR (CDCl3
) δ(ppm) 1.24 (t, 12H, J = 7.2 Hz), 3.58 (q, 8H, J = 7.2, 14.0 Hz), 5.89 (s, 1H), 6.42 (d, 2H, J = 16.0 Hz), 6.51 (d, 2H, J = 9.0 Hz), 7.66 (dd, 2H, J = 2.4, 9.0 Hz), 7.92 (d, 2H, J = 16.0 Hz), 8.34 (d, 2H, J = 5.0 Hz); 13
C NMR (CDCl3
) δ(ppm) 18.8, 42.7, 101.6, 106.3, 115.9, 118.6, 136.0, 144.2, 153.4, 159.8, 178.2.
A 1.0ml PBS solution of Aβ42 (250 nM) was added to each well of a 12-well plate, followed by the addition of a DMSO solution (10 μL) of CRANAD-3. The final concentrations of CRANAD-3 added were 50, 100, 250, 500, 750, and 1000 nM, with samples prepared in quadruplicate. The resulting plate was subjected to imaging using IVIS®Spectrum by setting Ex = 530nm, Em = 580nm to 840nm with 14 filters, subject height = 0.2cm, and FOV = C. Spectral unmixing was performed with LivingImage® 3.2 software by setting Component = 3 (representing 3 fluorophores that include autofluorescence, bound CRANAD-3 and free CRANAD-3), Photo Mask (this tool is for selecting the area of interesting), and Auto constraints (this tool is for constraining parameters to allow user zeroing low band pass and making non-negative contribution from fluorophores. In our experiments, we used the auto default model from the imaging system).
Tissue staining and imaging
Wild type (20-month old) or APP/PS1 (14-month old) mouse brain tissue was cut into 25-micron slices, fixed in 4 % formalin for 5 min and washed twice with PBS buffer. The tissue was incubated with 0.01% CRANAD-3 (in 50% ethanol) and washed. The tissue was subjected to imaging using the same parameter settings as were used for the phantom imaging.
Ex vivo brain imaging
A 24-month APP/PS1 mouse was intravenously injected with 1.5mg/kg CRANAD-3 (freshly prepared with 15% DMSO, 15% cremophor and 70% PBS buffer). After 1 hour, the mouse was sacrificed and the brain, heart, and spinal cord were collected. The three tissue/organs were subjected to imaging with Ex = 530nm, Em = 580 – 840nm, small binning, f = 2, FOV = B, and exposure time = 0.5s. Spectral unmixing was performed using LivingImage® 3.2 software by setting component = 3, Photo Mask, and Auto constraints.
In vivo imaging
Imaging was performed using an IVIS®Spectrum optical system (Hopkinton, MA). For dosage testing, APP-PS1 mice (n = 3) were shaved, and intravenously injected with 0.25, 0.5, 0.75, 1.0mg/kg of freshly prepared CRANAD-3 in 15% DMSO, 15% cremophor and 70% PBS. Fluorescence signals from the brain were recorded at 30 minutes after the injection of the probe. To monitor the progression of AD pathology (11-month and 19-month old APP-PS1 mice, n =5), image sequences were recorded at 10, 20, 30, 40, and 50 minutes post-injection. Imaging parameters were set at Ex = 570nm, Em = 620 – 840nm, medium binning, f = 4, FOV = C, and exposure time = 1s. Spectral unmixing was performed with LivingImage® 3.2 software by setting Component = 3, Photo Mask, and Auto constraints. To evaluate imaging results, an ROI was drawn around the brain region. For the bound signal, the ROI was drawn on the unmixed image corresponding to the bound spectrum, and total efficiency, which is fluorescence emission image normalized to the incident excitation intensity (radiance of the subject/illumination intensity), was used for quantification.