Nanoscale control of ligand display & release to probe barrier modulation, targeted internalization, and endosomal escape - ABSTRACT Biomolecular delivery is foundational to modern biology and medicine, offering precise control over cell behavior through surface receptor engagement and intracellular delivery of genes and proteins. Yet this potential is fundamentally limited by biological barriers, including tight junctions, plasma membranes, and endosomal compartments, that restrict transport, uptake, and cytosolic access. My lab specializes in modular biomaterial platforms for programmable ligand display and light-triggered intracellular release. We will leverage these precision tools to investigate how nanoscale features influence delivery mechanisms at the molecular and cellular scales. Theme 1: Can Nanoscale Engineering of Integrin Clustering Induce Transient Tight Junction Opening? Tight junctions are a major barrier to tissue-level delivery of large biologics, particularly across epithelial and endothelial interfaces. Previous work from our group demonstrated that nanotopographic cues remodel tight junctions via integrin signaling. Here, we hypothesize that spherical particles displaying nanoscale-clustered RGD ligands or integrin-targeting antibodies can recapitulate this remodeling effect without physical topography. Using our DNA-scaffolded ligand display system, we will systematically vary density, spacing, and flexibility to identify design rules for non-toxic, reversible tight junction modulation. This project will establish foundational principles for enhancing paracellular transport using spherical biomaterials. Theme 2: Can Ligand Architecture and Carrier Design Override Receptor-Intrinsic Internalization? While receptor- mediated endocytosis is often described as intrinsic to each receptor, growing evidence suggests that external presentation cues can redirect uptake pathways. We will apply our modular surface-engineering system to dissect how ligand architecture influences internalization for three distinct receptor systems: CD40 (clustering- driven), c-Kit (signal-dependent), and integrins (mechanosensitive). We hypothesize that tuning ligand density and flexibility on synthetic carriers can override receptor preferences, reprogramming trafficking and cargo fate. This project aims to uncover generalizable design strategies for targeted, receptor-agnostic intracellular delivery. Theme 3: Can NIR-Stimulated Nanoparticles Over Endosomal Barriers Across Cell Type and Cargos? Endosomal escape remains a major bottleneck for the intracellular delivery of therapeutic macromolecules. We previously demonstrated that hollow gold nanoshells enable siRNA release via NIR-triggered photothermal disruption. Here, we extend this strategy to mRNA and Cas9/gRNA delivery across challenging primary cell types, including primary T cells, macrophages, dendritic cells, and hematopoietic stem cells. We hypothesize that optimal escape thresholds depend on both cargo physicochemical properties and cell-intrinsic membrane repair capacity. By mapping delivery efficiency across cargo and cell types, we aim to define tunable parameters for safe and efficient endosomal release. Altogether, this program will generate broadly applicable principles for biologic delivery while training students across biomaterials, cellular analysis, and advanced imaging.
