A MULTI-FACETED APPROACH FOR ADDRESSING POOR DRUG TRANSPORT INTO THE LYMPH NODE

Abstract

The lymph node is a crucial component of the lymphatic system and the adaptive immune response. To carry out timely and efficient immune programming, the lymph node is comprised of multiple isolated lobules, or parenchyma regions, to host immune interactions. The isolation of the lobule is maintained by tightly controlled access from the fluid compartments, including blood vessels that significantly restrict passive transport and a fibrous barrier adjacent to the lymph sinuses that limits diffusive transport and excludes large particles. Although these entry points allow for efficient immune cell transit, free drug is largely excluded making the lymph node a pharmacological sanctuary site, or a location in which drug distribution is limited or excluded. Poor drug penetration into the lymph node lobule has been observed in numerous studies and is of great significance during the treatment of lymph node resident diseases, such as HIV and metastatic cancer. In HIV, the lymph nodes act as a long-lasting viral reservoir for patients receiving standard care therapy. While treatment methods have improved in recent decades, no sterilizing cure has been developed due to this pharmacological sanctuary site. In many metastatic cancers, the lymph node is the first site of tumor spread. While much of the pathology is left to be uncovered, lymph node metastases severely harm prognosis and aids in disease progression, with current therapeutic strategies often left ineffective. Our lack of insight on the transport phenomena occurring at these specialized boundaries limits our options. To make significant progress and move towards a viable treatment strategies for lymph node resident diseases, a two-step process is needed. First, using novel research techniques and animal models, we need to develop a better understanding of drug transport in the lymph node on a fundamental level and build tools to aid in this process. Second, innovative strategies for drug delivery and therapeutic design need to be employed to move past current insufficient gold standard treatments. We believe this dissertation has laid out a strategy to tackle both, using the synergy of experimental and computational methods. A primary obstacle in the understanding of drug transport in the lymph node is the lack of connections made between variability in lymph node geometry, drug biochemical properties, and the clinically seen heterogenous distribution of small molecules in the lobule. The ability to accurately produce 3-dimensional anatomical reconstructions of the lymph node could serve as a basis for quantitative morphological analysis, mapping of spatial drug distribution, and a better understanding of drug specific variations to transport. Therefore, we set out to build a novel tissue reconstruction pipeline to produce anatomical accurate 3D geometries of murine lymph nodes. Using custom sectioning methods, staining and imaging devices, and automated alignment and segmentation code, tissue compartments of interest were easily isolated and reconstructed in 3D. Using these 3D objects, spatial drug transport distributions could be modeled and predicted. Next, we set out to understand the degree to which cell-mediated transport contributed to overall antiretroviral (ARV) concentrations in the lymph node. Using evidence from spatial mass spectrometry modeling and uptake of ARV species by leukocytes, we hypothesized that for non-lipophilic species cell-mediated transport was a dominant mechanism. We developed a functional antibody blocking strategy to block lymphocyte ingress to test this mechanism. While some ARV species exhibited trends of cell-mediated transport, the contribution of this mechanism was smaller than originally predicted. From these findings, we also compared different drug classes and determined certain ARVs, such as islatravir and darunavir, displayed stronger lymph node accumulation than other species. This information can be used in the future to help design ARV species to target the lymph node reservoir. Lastly, leveraging mechanisms of lymphocyte ingress into the lymph node for therapeutic design could offer a solution to drug penetration issues. To that end we have developed a delivery strategy utilizing cryo-shocked T lymphocytes (CSTLs) as a cell mimetic and delivery vehicle. After cryoinjury, the CSTLs retain basic cellular functions, such as lymph node homing and transport across the high endothelial venules into the lobule, however, are permeable to load and release drug. We validated targeting and safety in vivo and confirmed strong lobule uptake with minimal immune responses. We next showed loading and release of a small molecule chemotherapeutic, cisplatin, which retained cytotoxic effect after release. Finally, using mass spectrometry techniques, we confirmed administration of cisplatin loaded CSTLs resulted in increased lymph node concentrations as compared to free drug. We believe this system serves as a backbone technology for many possible drug delivery applications. These studies lay the groundwork for a multi-faceted approach to address poor drug transport in the lymph node, and work to improve our potential for therapeutic design and treatment options in the future.