Nanoformulation-Based Approaches to Deliver Biologicals to Dendritic Cells and Liver Non- Parenchymal Cells
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Abstract
Liver non-parenchymal cells (NPCs), including Kupffer cells (KCs), dendritic cells (DCs), liver sinusoidal endothelial cells (LSECs), and hepatic stellate cells (HSCs), play a dual role in immunity. On the one hand, they exert tolerogenic activity under steady state conditions by default, while on the other hand, they are able to initiate adaptive immune responses. This may limit the efficacy of cancer immunotherapy, especially of vaccines targeting DCs in secondary lymphoid organs. Liver NPCs express numerous distinct pattern recognition receptors (PRRs), which are critical for pathogen detection, but also trap nanovaccines, that may be recognized by the immune systems as pathogens according to their size and non-self-surface properties. If a nanovaccine delivers antigen, this may risk antigen-specific tolerance. The active engagement of liver NPCs with antigen and adjuvant may dampen systemic anti-tumor T cell responses, highlighting the need for strategies that simultaneously activate DCs in secondary lymphoid organs and reprogram liver NPCs towards an immunostimulatory phenotype. The approaches taken in this thesis are visually summarized in the graphical abstract. First, methods for isolating liver NPCs, liver perfusion and liver dissociation, were established. These methods ensured reliability of the results regarding the activation states and responsiveness of liver NPCs to various well-defined molecular immunostimulatory adjuvants, such as Poly(I:C), R848, and cGAMP, which are frequently used in nanovaccine formulations. The effects of these adjuvants were analyzed in vitro as well as in vivo, simultaneously applied with mRNA lipid nanoparticles (LNPs), to analyze their effects on mRNA expression and immune cell activation. Furthermore, advanced nanocarrier systems were investigated to improve the delivery of antigens and adjuvants. This included the assessment of LNPs incorporating lipoamino xenopeptide carrier structures (LAF-Stp). The LAF-Stp units have a unique lipid architecture that is able to destabilize endosomal membranes and promote endosomal escape, thereby improving mRNA delivery and intracellular release. To refine immune cell targeting, trimannose- and TLR7/8 ligand-functionalized human serum albumin (TM-HSA) nanoparticles were designed, which target the DC-SIGN receptor and its homologues (e.g. LSIGN), on DCs and other liver NPCs. These nanoparticles demonstrated strong immune activation due to TLR7/8 agonist engagement, effectively stimulating cytokine production and enhancing antigen presentation of immune cells. In vivo studies showed efficient liver targeting, rapid clearance from the bloodstream, and robust immune activation. Further, to achieve transcriptional targeting, an optimized fascin1 gene-derived enhancer/promoter was used to drive DC-focused expression of DNA vaccines. Together, these efforts provide a foundation for the development for future immunotherapies that address the unique immunological environment of the liver while enhancing anti-tumor responses in secondary lymphoid organs.