ENGINEERING NANOPARTICLES FOR IMPROVED LYMPHATIC DELIVERY AND ELUCIDATING MECHANISMS REGULATING NANOPARTICLE TRANSPORT INTO LYMPHATICS

Abstract

Immune modulatory therapies usually need to be effectively delivered to lymph nodes to enhance therapeutic effectiveness. Lymphatic vessels exist throughout the body and can transport 10 – 250 nm therapeutic nanoparticles to lymph nodes, however, nanoparticle formulations required to maximize this transport, and the mechanisms governing this transport are poorly understood. Here, we probed the effect of surface charge, surface poly(ethylene glycol) (PEG) density, shape, and size on nanoparticle transport across LECs (LECs) and lymph node delivery. 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 non-PEGylated nanoparticles and that transport is maximized when the PEG is in a dense brush conformation corresponding to a high grafting density (Rf/D = 4.9). PEGylating 40 nm nanoparticles improved transport efficiency across LECs 68-fold compared to unmodified nanoparticles, demonstrating that the addition of PEG improves transport in a size-independent manner. We injected these nanoparticle formulations intradermally into C57Bl/6J mice and found that PEGylated 100 nm and 40 nm nanoparticles accumulate in lymph nodes within 4 hours, while unmodified nanoparticles accumulated minimally. Densely PEGylated nanoparticles also traveled furthest from the injection site. In this thesis, we also determined that nanoparticles are transported via both paracellular and transcellular mechanisms, and that both PEG conformation and nanoparticle size and shape modulates the cellular transport mechanisms. We also expanded our in-vitro lymphatic transport model to model important physiological conditions including transmural flow and found that the presence of this flow increased transport across lymphatic barriers in a shape and mechanism-dependent manner. To further investigate the mechanisms regulating nanoparticle transport, we generated a computational kinetic transport model that was able to quantify the contributions of both paracellular and transcellular transport mechanisms, as well as predict transport efficiency as a function of nanoparticle characteristics including size and surface chemistry. Using transport inhibitors, we can expand our system of equations to describe precise uptake and transport mechanisms, and the relation between nanoparticle formulation and mechanism. This computational model is one of the first to describe transport across lymphatic vessels, and offers some of the first definitions for coefficients used to quantitatively describe nanoparticles transport across LECs (i.e., permeability). Our computational, in-vitro, and in-vivo results indicate that nanoparticle surface charge, PEG conformation, and size are key criteria for nanoparticle design for effective lymphatic delivery with a dense, neutrally charged coating of PEG maximizing transport across LEC barriers and transport to lymph nodes. Optimizing nanoparticle formulation and surface characteristics, including PEG density, has the potential to enhance immunotherapeutic and vaccine outcomes.