Engineering a lifetime-based activatable probe for photoacoustic imaging

High-resolution, high-penetration depth activatable probes are needed for in-vivo imaging of enzyme activity. In this paper, we will describe the contrast mechanism of a new photoacoustic activatable probe that changes its excitation lifetime upon activation. The excitation decay of methylene blue (MB), a chromophore commonly used in therapeutic and diagnostic applications, is probed by photoacoustic lifetime contrast imaging (PLCI). The monomer of the dye presents a high-quantum yield of intersystem-crossing and long lifetime (70 μs) whereas the dimer is statically quenched with a short lifetime (a few ns). This forms the basis of a highly sensitive contrast mechanism between monomers and dimers. Two dimerization models - one using sodium sulfate, the other using sodium dodecyl sulfate - were applied to control the monomer-to-dimer ratio in MB solutions. Preliminary results show that the photoacoustic signal of a dimer solution is efficiently suppressed (< 20 dB) due to their short lifetime compared to the monomer sample. Flash-photolysis of the same solutions reveals a 99% decrease in transient absorption confirming PLCI results. This contrast mechanism can be applied to design a MB dual-labeled activatable probe bound by an enzyme-specific cleavable peptide linker. When the probe is cleaved by its target, MB molecules will separate by molecular diffusion and recover their long excitation lifetime enabling their detection by PLCI. Our long-term goal is to investigate enzyme-specific imaging in small animals and establish pre-clinical data for translational research and implementation of the technology in clinical applications.