Copper-Catalyzed Covalent Dimerization of Near-Infrared Fluorescent Cyanine Dyes: Synergistic Enhancement of Photoacoustic Signals for Molecular Imaging of Tumors.

Photoacoustic (PA) imaging relies on the absorption of light by chromophores to generate acoustic waves used to delineate tissue structures and physiology. Here, we demonstrate that Cu(II) efficiently catalyzes the dimerization of diverse near-infrared (NIR) cyanine molecules, including a peptide conjugate. NMR spectroscopy revealed a C-C covalent bond along the heptamethine chains, creating stable molecules under conditions such as a wide range of solvents and pH mediums. Dimerization achieved >90% fluorescence quenching, enhanced photostability, and increased PA signals by a factor of about 4 at equimolar concentrations compared to the monomers. In vivo study with a mouse cancer model revealed that dimerization enhanced tumor retention and PA signal, allowing cancer detection at doses where the monomers are less effective. While the dye dimers highlighted peritumoral blood vessels, the PA signal for dimeric tumor-targeting dye-peptide conjugate, LS301, was diffuse throughout the entire tumor mass. A combination of the ease of synthesis, diversity of molecules that are amenable to Cu(II)-catalyzed dimerization, and the high acoustic wave amplification by these stable dimeric small molecules ushers a new strategy to develop clinically translatable PA molecular amplifiers for the emerging field of molecular photoacoustic imaging.

Trafficking of a Single Photosensitizing Molecule to Different Intracellular Organelles Demonstrates Effective Hydroxyl Radical-Mediated Photodynamic Therapy in the Endoplasmic Reticulum.

Photodynamic therapy (PDT) is often used in preclinical and clinical treatment regimens. Reactive oxygen species (ROS) generated by photosensitizers (PSs) upon exposure to light induce cell death via diverse mechanisms. PSs can exert therapeutic effects in different cellular organelles, although the efficacy of organelle-specific PDT has yet to be determined as most previous studies use different PSs in different organelles. Here, we explored how a single PS, chlorin e6 (Ce6), targeted to different organelles altered the effectiveness of PDT. Ce6 intrinsically localizes to the ER after 4 h of incubation. Modification of Ce6 via conjugation with an octapeptide (LS765), a monosubstituted triphenylphosphonium (TPP) derivative (LS897), or a disubstituted TPP derivative (LS909) altered the intrinsic localization. We determined that LS765 and LS9897 predominantly accumulated in the lysosomes, but LS909 trafficked equally to both the mitochondria and the lysosomes. Moreover, the conjugation altered the type of ROS produced by Ce6, increasing the ratio of hydrogen peroxide to hydroxyl radicals. Irradiation of identical concentrations of the PSs in solution with 650 nm, 0.84 mW/cm2 light for 10 min showed that the TPP conjugates nearly doubled the hydrogen peroxide production from ∼0.2 µM for Ce6 and LS765 to ∼0.37 µM for LS897 and LS909. In contrast, Ce6 produced ∼1.5-fold higher hydroxyl radicals than its conjugates. To compare the effect of each PS on cell death, we normalized the intracellular concentration of each PS. Hydrogen peroxide-producing PSs are effective PDT agents in the lysosomes while the hydroxyl-generating PSs are very effective in the ER. Compared to the PSs that accumulated in the lysosomes, only the ER-targeted Ce6 exerted >50% cell death at either low light power or low intracellular concentration. By delineating the contributions of cellular organelles and types of ROS produced, our work suggests that targeting hydroxyl radical-producing PSs to the ER is an exciting strategy to improve the therapeutic outcome of PDT.