Photoacoustic tomography is a rapidly growing imaging modality that can provide images of high spatial resolution and high contrast at depths up to 5 cm. We report here the design, synthesis and evaluation of an activatable probe that shows great promise in enabling detection of the cleaved probe in the presence of the high levels of non-activated, un-cleaved probe, a difficult task to attain in absorbance-based modality. Before the cleavage by its target, proteolytic enzyme MMP-2, the probe, an activatable cell penetrating peptide, Ceeee[Ahx]PLGLAGrrrrrK, labeled with two chromophores, BHQ3 and Alexa750, shows photoacoustic signal of similar intensity at the two wavelengths corresponding to the absorption maxima of the chromophores, 675 and 750 nm. Subtraction of the images taken at these two wavelengths makes the probe effectively photoacoustically silent as the signals at these two wavelengths essentially cancel out. After the cleavage, the dye associated with the cell penetrating part of the probe(CPP), BHQ3, accumulates in the cells, while the other dye diffuses away, resulting in photoacoustic signal seen only at one of the wavelengths, 675 nm. The subtraction of the photoacoustic images at two wavelengths reveals the location of the cleaved (activated) probe. In the search for the chromophores that are best suited for photoacoustic imaging we have investigated photoacoustic signal of five chromophores absorbing in the NIR region. We have found that the photoacoustic signal did not correlate with the absorbance and fluorescence of the molecules, as the highest photoacoustic signal arose from the least absorbing quenchers BHQ3 and QXL 680.
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