The iridium(III)-cyanine complex (IrCy) was fabricated by conjugating an iridium(III) complex to a cyanine dye with an intense near-infrared (NIR) absorption. IrCy complex nanoparticles (NPs) with high water solubility and photostability were prepared by a solvent evaporation-induced self-assembly strategy. Considering their effective photacoustic (PA) imaging and generation of 1O2 property with 808 nm laser irradiation in aqueous solution, PA imaging guided NIR-driven photodynamic therapy in vivo was effectively conducted in the 4T1 xenograft model. We developed a real-time PA imaging methodology to investigate the pharmacokinetics, tumor targeting, and biodistribution of IrCy NPs. Taking advantage of the analysis of the PA signal of the common iliac vein, the blood circulation half-life of IrCy NPs in mice was calculated to be ∼18 h, and the enhanced permeability and retention effect of IrCy NPs offered the maximum targeting property in the tumor at about 24 h. The obvious change of PA imaging signal in kidney and bladder confirmed IrCy NPs should be excreted partially from the urine system, and the PA signal decreased from 12.5× to 2.8× in the liver, and from 28.8× to 9.4× in the spleen also confirmed the hepatic metabolic pathway.

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