Lepidium virginicum Water-Soluble Chlorophyll-Binding Protein with Chlorophyll A as a Novel Contrast Agent for Photoacoustic Imaging

Photoacoustic (PA) imaging (PAI) holds great promise for non-invasive biomedical diagnostics. However, the efficacy of current contrast agents is often limited by photobleaching, toxicity, and complex synthesis processes. In this study, we introduce a novel, biocompatible PAI contrast agent: a recombinant water-soluble chlorophyll-binding protein (WSCP) from <i>Lepidium virginicum</i> (LvP) reconstituted with chlorophyll a (LvP-chla). LvP-chla exhibits a strong and narrow absorption peak at 665 nm, with a molar extinction coefficient substantially higher than oxyhemoglobin and deoxyhemoglobin, enabling robust signal generation orthogonal to endogenous chromophores. Phantom studies confirmed a linear relationship between PA signal amplitude and LvP-chla concentration, demonstrating its stability and reliability. In vitro cytotoxicity testing using 4T1 cells showed high cell viability at 5 mg/mL, justifying its use for in vivo studies. In vivo experiments with a 4T1 tumor-bearing mouse model demonstrated successful tumor localization following intratumoral injection of LvP-chla, with clear visualization via spectroscopic differentiation from endogenous absorbers at 665 nm and 685 nm. Toxicity assessments, both in vitro and in vivo, revealed no adverse effects, and clearance studies confirmed minimal retention after 96 h. These findings show that LvP-chla is a promising contrast agent that enhances PAI capabilities through its straightforward synthesis, stability, and biocompatibility.