The sensitivity of the PAI technique to image deeply situated tumors can be increased drastically by utilizing exogenous contrast agents. The NIR-absorbing dyes, such as IRDye800CW [31
], AlexaFluor 750 [37
] and indocyanine green (ICG) [38
], have been used to enhance PA contrast. However, among the exogenous contrast agents, AuNPs have attracted attention in nanoparticle-based PAI owing to their unique optical properties from the surface plasmon resonance (SPR) effect. Because of the SPR effect, AuNPs have an absorbance that is orders of magnitude higher than NIR dyes. For example, gold nanospheres, nanorods, nanoshells, nanocages and nanobeacons have been used in PAI because of their tunable and strong longitudinal plasmon resonance in the NIR [39
]. The cytotoxicity of these nanoparticles is debatable and often emerges in a dose- and time-dependent manner for different types of nanoparticles; hence, further investigation into AuNPs toxicity is necessary [47
]. The details on toxicity and pharmacodynamics of nanoparticles are beyond the scope of the review and are therefore not discussed here.
By attaching targeting moieties to exogenous agents, specific molecular information regarding the tumors can be obtained. AuNPs with different optical absorption properties can be conjugated to cancer-specific biomarkers, such as growth factor receptors and integrins (). By utilizing multiple targeted AuNPs, multiplex molecular labeling of a tumor can be achieved and multi-wavelength PAI can image the heterogenous accumulation and interaction of AuNPs with cancer cells in vivo
]. Fluorescent optical probes, such as quantum dots, also provide PA contrast and can be used for multiplex labeling of tumors [48
Exogenous PA contrast agents used for detecting tumors in vivo
There have been several advances in molecular PAI that help describe crucial functional and molecular interactions between tumor cells and the surrounding micro-environment. For example, the feasibility of utilizing a multi-wavelength PAI technique to monitor molecular interactions of epithelial growth factor receptor (EGFR)-targeted AuNPs in 3D tissue cultures and ex vivo
tissue has been evaluated [44
]. Briefly, AuNPs (spheres of 50 nm diameter) functionalized with antibodies bind to EGFR. This specific targeting of AuNPs to EGFR causes plasmon resonance coupling between adjacent nanoparticles and changes their absorbance spectra so that it can be detected as a change in the PA signal amplitude. Overall, the results indicate that PAI together with bioconjugated AuNPs have the potential to image nano-molecular interactions. Because of the concerns that nano-scale agents might cause long-term toxicity in vivo
, biodegradable gold nanoclusters have been developed as a contrast agent [54
]. The size of these biodegradable nanoclusters, consisting of sub-5-nm AuNPs and a biodegradable polymer binder, is less than 100 nm. The nanoclusters are also pH-sensitive and will biodegrade in the acidic environment of the endosome. After degradation, the 5-nm AuNPs are excreted out of the body, thereby preventing toxic accumulations.
Another recent advancement in PAI is the use of photo-activable probes to provide a target-dependent photoacoustic signal and they show superior specificity and sensitivity as compared to the probes that donot interact with the target. For example, photo-activable probes have been designed to specifically target matrix metalloprotease 2 (MMP-2), a protease found to be overexpressed in many aggressive cancers[56
]. The probe’s peptide platform consists of an activatable cell-penetrating peptide (ACPP) that is recognizable by MMP-2, in both in vitro
and mouse models. Before cleavage by MMP-2, the intact probe shows PA signals of similar intensity at the two wavelengths corresponding to the absorption maxima of the chromophores BHQ3 (675 nm) and AlexaFluor 750 (750 nm). When the probe is cleaved by the appropriate enzyme, the BHQ3 dye associated with the CPP portion of the probe accumulates in the nearby cells, while the Alexa dye diffuses away. This results in a PA signal visible only at 675 nm [56
Recently, emphasis has been placed on multi-modal nanosystems that can enhance contrast in two or more imaging modalities, including microbubbles [57
], perfluorocarbon-based nanobubbles [58
], and nanowontons [59
]. For example, nanowontons consist of ferromagnetic (cobalt core) core coated with gold for biocompatibility and a unique shape that enables optical absorption over a broad range of frequencies; the magnetic core acts as an MRI contrast agent and the gold coating provides the optical absorption contrast for PAI [59
]. With the evolution of combined imaging strategies, these multi-modal nanostructures will play a prominent role in cancer detection and treatment.