Alice Comberlato
Recent advances in the field of nanomaterials demonstrated how their use for vaccination can drastically improve immune responses, mainly by enhancing delivery and uptake of vaccine components. However, our understanding of the connections between presentation of immune-modulatory agents on nanomaterials and cellular responses is still very limited. DNA-based nanostructures evolved as an ideal platform to display ligands with unprecedented nanoscale precision for the investigation and control of ligand-receptor interactions involved in cellular activation, being at the same time a carrier for these therapeutics. With this work, we aim to set the next steps for DNA nanoparticles to become functional structures in medicine and to develop DNA-based nanomaterials to study aspects of the complexity of our immune system.In Chapter 1, the latest developments and key features of immune-engineering nanomaterials for vaccination with nucleic acid adjuvants are summarized. Moreover, we report the importance of ligand spacing as new parameter in multivalent systems for immune modulation and how DNA-based nanomaterials demonstrated the significance of ligand patterns for regulated cellular responses.In Chapter 2, we developed a DNA origami disk-shaped nanoparticle with nanoscale control on ligand presentation, to be used as platform for cellular interaction studies at the single-molecule level. By DNA-PAINT super resolution microscopy, we verified spatial patterns on our nanoparticle, showing that our in silico design matches at the one-nanometer-level with experimental measurements. Additionally, we verified that the oligolysine-PEG protective coating does not hamper the accessibility of functional sites. In Chapter 3, we present a systematic analysis of the effects of surface parameters of DNA-based nanoparticles on cellular uptake. We demonstrated the influence of surface charge, stabilizing coating, fluorophore types, functionalization technique, and particle concentration on material uptake in three different cell models: tumor cells, macrophages, and dendritic cells. Additionally, we showed approaches to reduce the risk of data artifacts in vitro. In Chapter 4, we employed our DNA platform to demonstrate the importance of controlling the spacing of adjuvant presentation on nanoparticles for the activation of immune cells. Exploiting pre-existing crystallography data, we created DNA disk nanoparticles presenting CpG-motif adjuvants in rationally designed spatial patterns to activate Toll-like Receptor 9. We showed that stronger immune activation is achieved when CpG ligands are matching the distance of the binding sites of the active dimer form of the receptor, compared to random presentation or free form of CpG at equal dose. Moreover, we show how linkers for conjugating active molecules to nanomaterials alter the spatial tolerance of binding and cell activation levels. Taken together, our results show that proper receptor targeting and engineering surface parameters for uptake of nanomaterials would help to develop potent and specific immune therapies. The rationally designed and spatially controlled presentation of therapeutics would aid future vaccine strategies to manipulate the immune system at the nanoscale level and to improve dose sparing, limiting toxicity often associated with the use of high doses of drugs.
EPFL2022
