Nanoparticles with dense poly(ethylene glycol) coatings with near neutral charge are maximally transported across lymphatics and to the lymph nodes.

Lymphatic vessels have recently been shown to effectively deliver immune modulatory therapies to the lymph nodes, which enhances their therapeutic efficacy. Prior work has shown that lymphatics transport 10-250 nm nanoparticles from peripheral tissues to the lymph node. However, the surface chemistry required to maximize this transport is poorly understood. Here, we determined the effect of surface poly(ethylene glycol) (PEG) density and size on nanoparticle transport across lymphatic endothelial cells (LECs) by differentially PEGylated model polystyrene nanoparticles. Using an established in-vitro lymphatic transport model, we found PEGylation improved the transport of 100 and 40 nm nanoparticles across LECs 50-fold compared to the unmodified nanoparticles and that transport is maximized when the PEG is in a dense brush conformation or high grafting density (Rf/D = 4.9). We also determined that these trends are not size-dependent. PEGylating 40 nm nanoparticles improved transport efficiency across LECs 68-fold compared to unmodified nanoparticles. We also found that PEGylated 100 nm and 40 nm nanoparticles accumulate in lymph nodes within 4 h after intradermal injection, while unmodified nanoparticles accumulated minimally. Densely PEGylated nanoparticles traveled the furthest distance from the injection site and densely PEGylated 40 nm nanoparticles had maximum accumulation in the lymph nodes compared to low density PEGylated and unmodified nanoparticles. Finally, we determined that nanoparticles are transported via both paracellular and transcellular mechanisms, and that PEG conformation modulates the cellular transport mechanisms. Our results suggest that PEG conformation is crucial to maximize nanoparticle transport across LECs and into lymphatic vessels, making PEG density a crucial design. Optimizing PEG density on nanoparticle formulations has the potential to enhance immunotherapeutic and vaccine outcomes. STATEMENT OF SIGNIFICANCE: Lymphatic vessels are an emerging target for drug delivery both in the context of modulating immune responses and enhancing bioavailability by avoiding first pass hepatic metabolism after oral delivery. Lymphatic vessels are the natural conduits from peripheral tissues to the lymph nodes, where the adaptive immune response is shaped, and eventually to systemic circulation via the thoracic duct. Lymphatics can be targeted via nanoparticles, but the surface chemistry required to maximize nanoparticle transport by lymphatics vessels remains poorly understood. Here, we demonstrate that coating nanoparticles with hydrophilic polyethylene glycol (PEG) effectively enhances their transport across lymphatic endothelial cells in vitro and in vivo and that both paracellular and micropinocytosis mechanisms underly this transport. We found that dense PEG coatings maximize lymphatic transport of nanoparticles, thus providing new material design criteria for lymphatic targeted drug delivery.