PROGRAM | Chemistry & Biochemistry

By: Allyson Dang Chair: Jason Gleghorn

ABSTRACT

Selective therapeutic delivery to the lymph node (LN) is of great challenge that has the potential to address a variety of unmet clinical needs. The lymph node is a crucial component of the lymphatic system and the adaptive immune response. In order to carry out efficient immune programming, the LN is compartmentalized into highly isolated cell-rich lobules. Consequently, the LN anatomy and lymph membrane barriers pose significant transport limitations in preventing delivery of therapeutics to their intended target, resulting in poor transport of free drug into lymphoid tissue. This inadequate drug penetration is of great significance during the treatment of lymph node resident diseases such as HIV. In HIV, latently infected cells can evade antiretroviral (ARV) drugs and immune responses for decades, and patients with fully suppressed viremia have presented with high levels of free virus in the LN. The presence of persistent virus coupled with poor drug penetration implies that viral replication has not been completely suppressed, implicating LNs as hubs for viral recrudescence consisting of latently infected cells^1–3. The mechanisms of transport into the LN are significant for therapeutic design, however many aspects of transport across the LN boundaries remain unclear.

Improved therapeutic strategies that target viral reservoirs, or “pharmacological sanctuary sites”, such as the lymph node could create substantial progress towards treating LN resident diseases. Current studies have developed particle-based therapeutic strategies for localized drug delivery to the lymph node. While an improvement compared free drug delivery, these delivery platforms are nanoparticles (NPs) primarily developed for cancer treatment via subcutaneous administration by direct internodal injection. These systems significantly rely on accessible, superficial draining LNs and transport through small fenestrations in the interconnected lymphatic sinus network for access to the lymphatic system, and ultimately, can only target a fraction of the lymph nodes that make up the entire lymphatic system. Additionally, the LN’s specialized biological barriers remain a challenge for therapeutics from penetrating the cell-rich regions of the lymph node. By advancing on these approaches and designing particle-based platforms that can be delivered intravenously, the whole lymphatic system becomes accessible.

Within the past decade, the advent of biomimetic therapeutic platforms has revolutionized particle-based drug delivery. Specifically, membrane-wrapped nanoparticles (MWNPs) have been at the forefront of this field. Cell membrane coating has been utilized to address transport limitations for NP based platforms and has been demonstrated to extend circulation time and provide site-specific accumulation and enhance nanoparticle performance in a variety of contexts across a broad spectrum of applications. However, the nano size scale of these delivery systems is susceptible to rapid clearance by the mononuclear phagocyte system in the bloodstream. Microparticles (MPs), on the other hand, are more comparable in size to that of circulating cells. T-lymphocytes possess intrinsic capability to bypass the biological barriers that isolate the LN and extravasate across vasculature into the cell-rich lobules of the LN. Membrane-wrapped microparticles (MWMPs) are advantageous in that they host the dual potential in being both bio- and cell specific-mimetic. When developed into an immune cell-mimetic drug delivery carrier, this “Trojan horse” inspired T-lymphocyte mimetic MWMP may be introduced systemically to bypass immune detection and enhance targeting and localization capability to lymphoid tissue.

This dissertation is divided into three main aims to understand drug transport and enhance delivery to the LN.

In aim 1, we investigated the contributions of T lymphocyte-mediated drug transport into the LN. Circulating T-lymphocytes are recruited to LNs and known to migrate throughout the lobule for extended periods of time. Thus, lymphocytes can act as drug carriers and shuttle intracellular cargo through the lymph node incidentally during trafficking. We hypothesized that functional antibody blocking inhibiting T-lymphocyte entry to the LN would result in a knockdown of ARV concentration in lymphoid tissue. However, it is difficult to quantify the concentration of ARV drug delivery to different LN compartments. Here, we developed a strategy to fractionate the lymph node into intracellular and extracellular compartments to elucidate the contributions of drug delivery by lymphocyte ‘backpacking’ into lymphoid tissue. Next, we extracted ARV in each fraction and quantified intra and extracellular LN ARV concentrations related to the plasma concentration by conventional mass spectrometry.

In aim 2, we created an injectable hydrogel platform to create microgel depots for sustained, localized delivery. Hydrogel MPs enable the encapsulation of biotherapeutics. However, depending on the intended therapeutic, fabrication of structurally and compositionally homogenous microgels can be challenging, which is of concern for drug delivery. To match the success of bulk hydrogel loading, we leveraged dynamic crosslinking to create an injectable hydrogel amenable with microfluidic platforms to fabricate homogenous hydrogel MPs.

In aim 3, we developed a strategy to fabricate membrane-wrapped alginate hydrogel MPs. We enriched T-lymphocyte plasma membrane by hypotonic lysis followed by mechanical homogenization. We then used conventional microfluidic techniques to fabricate alginate MP cores representative in size of LN resident cells (5-25 µm).  Lastly, we coextruded the MP and membrane components to manufacture T-lymphocyte derived membrane-wrapped MPs. We characterized the components to fabricate MWMP and lay the foundation to translate this platform in vivo.

In summary, the work in this dissertation includes the analysis of transport mechanisms into the LN to develop microparticle-based delivery platforms for enhanced localization and sustained drug delivery to the lymph node.

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