Quinone-fused porphyrins as contrast agents for photoacoustic imaging

Naphthoquinone fused porphyrins showed higher photoacoustic signals than ‘standard’ indocyanine green (ICG). In this context, the insertion of Zn(ii) resulted in the most potent photoacoustic dye, which also proved to be biocompatible and stable in serum.


General.
All the reactions, work-up and chromatography were performed under protection from light (wrapping in alumina foil). All the glassware for the reactions was oven dried at 100 °C and cooled under high vacuum (HV) before use and kept under argon. Absolute solvents were prepared by distillation over calcium hydride. Organic solutions were concentrated under reduced pressure on a Büchi rotary evaporator (< 37°C water bath). Thin-layer chromatography was carried out using Merck Kieselgel 60 F254 (230-400 mesh, 25x25cm for preparative scale).

Photoacoustic Imaging:
The pre-clinical PA device VEVO LAZR (from Visualsonics Inc, Amsterdam, NL) equipped with a LZ 250 transducer having a centre frequency of 21 MHz was used for scanning (26 db gain). The VEVO LAZR built-in pulse laser (wavelengths between 680-970 nm) was warmed up for 30 min before use and energy was calibrated using an internal sensor. Tube phantom experiments were carried at 9-11 mm depth in a water chamber with total of 59 (for 5 nm step) or 146 frames (2 nm steps). The Vevo 2100 software was used for data processing. During the measurements of serial dilutions, all variable parameters were kept constant i.e. photoacoustic gain, laser power, focus depth, frame averaging, and frame rate.

Calculation of relative photoacoustic intensity:
PA intensity (a.u) was measured in tube phantoms for all the solutions using identical regions of interest. The PA Avg signal/conc ratios of the black porphyrins were divided by the according ratio of ICG (then, for ICG relative PA intensity becomes 1).

Cell Viability XTT assay
In a 96-well plate, A549 cells (5x10 3 to 10x10 3 ) were seeded and treated with DMEM media supplemented with 10% FCS, 1% pencillin/streptomycin (P/S). The cells were incubated at 37 °C (in 5% CO 2 incubator) for 24 h to let the cells attach to the surface of the 96-well plate. The substance in DMEM media (conc. 0.001 µmol/ml to 0.1 µmol/ml) was added to the above well plate, and further incubated at 37 °C in 5% CO 2 incubator for 24 h. XTT-solution was freshly prepared by XTT test kit (using the prescribed protocol from Gibco), and 50 µL of this mixture was added to each well of the above cells. After 2-4 h, the absorption was measured in TECANreader at 475 nm wavelength using 660 nm as reference.
Female nude mice (4-6 weeks, n = 6, performed according to the Guidelines for the Care and Use of Animals for Research, approved by MSKCC's Institutional Animal Care and Use Committee) were intravenously injected with 3-Zn (150 μL, 100 μM, 30% PEG300 in PBS, n=3) and PBS (150 μL, n=3). At 1 h post injection, mice were euthanized by asphyxiation with CO 2 .
Blood was collected via cardiac puncture and selected organs, liver, kidney, and muscle, were harvested for ex vivo MSOT imaging. Wavelengths from 700 nm to 900 nm in 20 nm steps (power: 100 mW) were used for excitation, with 5 acquisitions averaged per wavelength per frame. After image reconstruction, linear spectral unmixing was conducted to detect and select the specific signal of 'black' porphyrin from other intrinsic signal such as oxygenated and deoxygenated hemoglobin. All images were scaled to the same threshold (arbitrary units) to compare tissue injected with black porphyrin from PBS. To quantify the signal, the black porphyrin channel (green) was averaged over a circular area at the slices central to each tissue.
Reaction progress was followed through UV/Vis and TLC, after 1.5h the flask was removed from oil bath to cool down the reaction to room temperature. The resulting dark suspension washed with sat aq. NaHCO 3 (3 x 15 mL) and product mixture extracted into CH 2 Cl 2 (ca. 4 x 10 mL) until the organic extracts were colourless. The combined organic extracts were filtered
Resulting mixture washed with acidic water (pH 2, 3 x 15 mL) and product extracted into