(M1230-08-48) Antibody-Targeted Liposomes Encapsulating Indocyanine Green Aggregates for Photoacoustic Imaging (PAI) and Image-Guided Drug Delivery

Purpose: Photoacoustic imaging (PAI) is an emerging technique that combines optical and ultrasound imaging to provide high-resolution, deep-tissue visualization, making it highly valuable for cancer diagnosis. Indocyanine green (ICG), an FDA-approved near-infrared dye, is widely used as a contrast agent in PAI. However, free ICG has significant limitations, including rapid clearance from the body and weak signal output. These challenges can be addressed by forming ICG J-aggregates, which offer stronger and red-shifted absorption signals near 895 nm. Yet, these aggregates are typically unstable in water and difficult to encapsulate effectively within delivery systems. This study aims to develop a liposome-based platform for improved photoacoustic imaging (PAI) and potential image-guided drug delivery. The goal is to achieve stable encapsulation of indocyanine green (ICG) aggregates using an active loading approach that applies a pH gradient and controlled heat treatment. This method is intended to address the challenge of poor retention and instability of ICG aggregates in aqueous environments. The platform is also designed to allow surface modification with targeting ligands in order to enhance imaging specificity. Additionally, it may support co-loading of therapeutic agents, offering combined diagnostic and therapeutic functionality for cancer applications.

Methods: We used an active loading method based on an ammonium sulfate gradient to encapsulate ICG into liposomes. A transmembrane pH gradient was established to facilitate dye loading, and free dyes were removed using centrifugal filters. The liposomes were then heated at 55°C for 48 hours to promote the formation of ICG aggregates within the vesicles. Particle size and distribution were measured using dynamic light scattering (DLS), and UV-Vis spectroscopy was used to confirm ICG aggregation by identifying red-shifted absorbance peaks. We are currently working on the directional conjugation of anti-EGFR antibodies to the liposome surface. Antibody binding will be verified using ELISA, and flow cytometry and photoacoustic imaging will be used to assess targeting efficiency in EGFR-positive cancer cells.

Results: Liposomes were successfully formed and actively loaded with ICG using a pH-gradient method. Heating the liposomes at 55°C for 48 hours resulted in the formation of stable ICG aggregates within the vesicles. Dynamic light scattering (DLS) showed a uniform particle size of 146 ± 5 nm, indicating suitability for in vivo applications. UV-Vis spectroscopy confirmed the presence of ICG aggregates with a distinct red-shifted absorbance peak around 895 nm, demonstrating successful encapsulation and aggregate formation.

Conclusion: We developed a liposome-based system that encapsulates stable ICG aggregates. The liposomes showed appropriate size and strong optical properties for photoacoustic imaging. We are currently preparing EGFR-targeted liposomes and will evaluate their targeting capabilities using ELISA, flow cytometry, and photoacoustic imaging. This delivery platform has strong potential for use in targeted cancer imaging. We will also investigate co-loading with anticancer drugs to support future image-guided therapeutic applications.