Near-Infrared Fluorescence Imaging and Photothermal Therapy of Tumors Using a Targeted Helical Protein Scaffold.

Photothermal therapy (PTT) using near-infrared (NIR) photoactive agents offers a minimally invasive approach for tumor ablation; however, many existing platforms rely on polymeric or nanoparticle carriers that limit targeting precision and control over dye loading. Here, we report the design of a modular eight-helix protein scaffold containing 20 cysteine residues that enables covalent, site-specific conjugation of the NIR dye AQuora800-maleimide. The protein scaffold is expressed with different HER2-targeting ligands, and in vitro screening identifies an affibody dimer-based construct (AD-20AQ) as the most effective platform for targeted fluorescent imaging and therapy. In vivo pharmacokinetic imaging studies in a 4T1 murine tumor model reveal enhanced tumor accumulation of AD-20AQ compared to a monolabeled affibody dimer (AD-1AQ), with sustained retention in HER2-positive tumors. Furthermore, PTT of HER2 tumors with an 808 nm laser, following intravenous administration of AD-20AQ, leads to complete tumor ablation. Collectively, these results establish targeted, labeled protein scaffolds as a versatile platform for NIR fluorescence imaging and photothermal therapy.