Fibronectin Enriched Protein Corona for Enhanced Tumor Targeting of Nanoparticles - Project Summary Nanomedicine holds great promise for transforming disease treatment, yet its clinical translation is limited by suboptimal targeting efficiency and unpredictable interactions with biological systems. Upon exposure to biological fluids, nanoparticles rapidly acquire a protein corona (PC) that defines their biological identity and governs circulation, biodistribution, and cellular interactions. While most studies have focused on inorganic nanoparticles, there remains a critical knowledge gap regarding how polymeric nanocarriers interface with biological environments. Understanding this relationship is essential for elucidating the mechanisms that regulate cellular transport and biodistribution and for designing more precise and effective delivery systems. We discovered that PAZA, an ultra-small, drug-conjugated polymeric nanoparticle, forms a fibronectin (FN)- enriched corona that binds integrin á5â1 (ITGA5) on tumor endothelium and tumor cells, enabling deep tumor penetration and selective accumulation. This project will define how FN coronas form and function, how they remodel during circulation, and how nanoparticle design can be leveraged to recruit FN and drive transcytosis. Aim 1 will dissect the relative contributions of transcytosis versus passive EPR using in vitro endothelial transport assays, a Zombie mouse model, and ITGA5-deficient tumors and mice, while also identifying additional FN-ITGA5–high tumor types through bioinformatics and validating them using qRT-PCR, IHC, and functional models such as microvessel-on-a-chip, Transwells, and 3D organoids. In vitro assays provide mechanistic insights, but cannot recapitulate complex factors such as circulation dynamics, protein corona evolution, immune clearance, and tumor vasculature heterogeneity. Use of syngeneic tumor-bearing mice (LLC in C57BL/6 background) is required to study PAZA delivery in an intact immune system, while genetically engineered endothelial ITGA5 knockout mice are necessary to directly test mechanistic contributions of ITGA5- mediated transcytosis. The Zombie model is critical to differentiate EPR versus active transport in vivo. Aim 2 will determine whether FN alone is sufficient for targeting by comparing pre-coated PAZA with in situ-formed coronas, with time-resolved in vivo profiling and shear-flow studies assessing FN stability, competitive exchange with serum proteins, and effects on circulation and biodistribution. Aim 3 will establish design rules by systematically varying nanoparticle size and surface chemistry, combining label-free proteomics in mouse and human serum with molecular modeling to map FN-polymer interactions and predict corona stability. This work will generate foundational insights into how protein coronas regulate nanoparticle behavior and establish a new ligand-free strategy to enhance tumor targeting, thereby advancing the clinical translation of nanomedicine.
