Biomedical imaging has been revolutionized by the field of molecular imaging that offers the possibility of understanding diseases at the molecular level1,2
. Photoacoustic tomography, a rapidly growing imaging technique, combines optical and ultrasound imaging in such a way that the result is a modality with characteristics superior to each of the component imaging techniques3,4
. As a molecular imaging modality that offers both high spatial resolution and high contrast, photoacoustic tomography utilizes endogenous4,5
as well as exogenous6–8
light absorbers as entities providing the optical contrast in biological tissues. However, probes that show signal only in the presence of a specific target, so called activatable or smart probes, have not yet been reported for photoacoustic imaging. Activatable probes for optical and magnetic resonance imaging have been extensively studied and applied for in vivo
. They show superior specificity and sensitivity to the probes that provide signal regardless of interaction of probe with the target. Here, we wish to report the design and evaluation of a photoacoustic smart probe that provides a target dependant photoacoustic signal and enables visualization of the signal only in the presence of the target of interest.
Dual and multi-wavelength photoacoustic imaging have been employed in acquiring impressive images with clearly distinguished endogenous molecule-specific signals15–18
. We designed our probes wanting to take advantage of dual wavelength imaging (). In the intact state, the probe should show photoacoustic signal at the two wavelengths that correspond to the absorption maxima of the two chromophores within the probe. When the probe is cleaved by the appropriate enzyme, the dye associated with the cell penetrating part of the probe (CPP) accumulates in nearby cells, while the other dye component diffuses away. Photoacoustic signal is thus expected only at the absorption wavelength of the dye accumulated in the cells. The main criteria for choosing the chromophores were high absorption in the near infra-red (NIR) region and well separated, mutually non-overlapping, absorption spectra. The probes were designed to be specific for an extensively studied target, matrix metalloprotease 2 (MMP-2), a protease found to be over-expressed in many cancers and associated with tumor aggressiveness19,20
. Activatable cell penetrating peptide (ACPP) was selected as the probes' peptide platform because of its proven efficacy in detecting MMP-2, both in vitro
and in mouse models21,22
. ACPP has the MMP-2 cleavable amino acid sequence between polyarginine based cationic (CPP) and polyanionic domains. We hypothesized that the hairpin structure22
of ACPPs would allow the interaction between the two dyes resulting in either resonance energy transfer or static quenching, both of which could lead to a target-dependant photoacoustic probe. The peptide sequence used in our study, Ceeee[Ahx]PLGLAGrrrrrK, differs from the one reported by Jiang et. al.22
in the number of arginine and glutamic acid residues. The number of these amino acids is important for the transduction efficiency of the polyarginine sequence23,24
. We have chosen the shortest polyarginine sequence shown to provide efficient cargo delivery to facilitate the separation of the charged parts of the peptide after enzymatic cleavage.
Figure 1 Scheme illustrating probe design. In its intact state, the probe produces a photoacoustic signal at two wavelengths (λC1, λC2) corresponding to the absorption maxima of the two chromophores C1 and C2. When cleaved by the appropriate enzyme, (more ...)