Development of fluorinated dyes for deeper tissue photoacoustic imaging with phase changing nanodroplets

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Abstract. Photoacoustic imaging (PAI) is a promising modality that is non-ionizing, low-cost, and offers high- contrast and high-spatiotemporal-resolution imaging in a platform that is amenable for high-throughput preclinical use and for specific clinical applications. However, widespread use of molecular PAI is severely limited by availability of validated contrast agents. Currently available contrast agents either do not have adequate photostability under the pulsed illumination that is required for PAI, lack sufficient PAI-signal-generation ability for deep imaging, or their absorbance spectra significantly overlap with those of hemoglobin, which reduces imaging sensitivity. In order to address these limitations, a new class of PAI contrast agents was proposed that is based on phase-changing perfluorocarbon (PFC) nanodroplets (NDs). These agents are based on a liquid PFC core and a light-absorbing “fuse” in the form of a dye or a nanoparticle. Illumination of these NDs with a pulsed laser triggers liquid-to-gas transition of the PFC core heated by light-absorbing chromophores that results in a very strong PAI signal. Therefore, these agents are often referred to as Laser-Activated NDs (LANDs). Furthermore, after laser excitation PFC microbubbles can re-condense back into their liquid nanodroplet form, which can allow multiple excitations and the possibility for dynamic imaging contrast and super-resolution PAI. However, evaluation of this exciting contrast agent design by multiple research groups revealed one critical limitation – commonly used dye molecules or nanoparticles are not soluble or mixable with perfluorocarbons. Therefore, current LANDs contain their “fuses” (i.e., dye absorbers) in the shell with a loading efficiency and distribution of the dyes that is highly variable depending on specifics of a LAND's coating and a dye's chemical structure. Importantly, in addition to the limitations associated with irreproducibility and a shot shelf-life of LANDs due to leakage of dye molecules from a LAND's shell, recent studies of phase-changing NDs showed the advantage of heating LANDs from within the core for an effective liquid-to-gas transition. These data underline the importance of heating inside an ND's core for activation of LANDs that cannot be effectively achieved with peripherally located chromophores. Here we propose to address weaknesses of the prior research by developing fluorinated dyes with absorbance in the first and second near-infrared tissue windows (NIR-I and NIR-II). Our hypothesis is that the fluorinated dyes will be soluble inside the PFC core, thus resulting in highly reproducible, stable LAND formulations with greatly improved laser activation efficacy. To reflect these advancements in LAND formulation, we refer to PFC NDs doped with fluorinated dyes as enhanced LANDs (eLANDs). We posit that an increase in concentration of uniformly distributed fluorinated dyes inside the PFC core will dramatically improve efficacy of eLAND's activation. Our estimates show that this gain in activation efficacy could be associated with a highly significant (on the order of centimeters) increase in depth sensitivity of PAI with eLANDs; such increase in depth penetration could be a game changer in molecular PAI in pre-clinical and clinical settings.