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

## TL;DR

A new biocompatible contrast agent for photoacoustic imaging is developed from a plant protein and chlorophyll a, offering strong imaging performance and safety.

## Contribution

A novel water-soluble chlorophyll-binding protein reconstituted with chlorophyll a is introduced as a stable and biocompatible contrast agent for photoacoustic imaging.

## Key findings

- LvP-chla shows a high molar extinction coefficient and linear PA signal generation with a penetration depth of 6.30 mm in tumor visualization.
- In vitro and in vivo tests confirm high cell viability and no adverse effects, with clearance within 96 hours.
- LvP-chla enables spectroscopic differentiation from endogenous absorbers at 665 nm and 685 nm in tumor imaging.

## Abstract

What are the main findings?

LvP-chla exhibits a high molar extinction coefficient (59,716 M−1cm−1 at 665 nm), linear PA signal generation, and effective tumor visualization in a 4T1 mouse model with a penetration depth of 6.30 mm.

In vitro cytotoxicity testing showed high cell viability at 5 mg/mL with no in vivo adverse effects, and clearance within 96 h, confirming biocompatibility.

What is the implication of the main finding?

LvP-chla offers a stable and biocompatible contrast agent for PAI.

The straightforward synthesis and efficient clearance of LvP-chla make it a promising candidate for clinical translation in non-invasive diagnostics.

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 Lepidium virginicum (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.