DNA-based biomaterials for nanoscale controlled modulation of immune cells

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.

DNA origami nanostructures for controlled therapeutic drug delivery

Maartje Martina Cornelia Bastings, Jorieke Weiden

DNA nanostructures are emerging as a versatile platform for controlled drug delivery as a result of recent progress in production yield and strategies to obtain prolonged stability in biological environments. The construction of nanostructures from this unique biomaterial provides unparalleled control over structural and functional parameters. Recent applications of DNA origami-based nanocarriers for therapeutic drug delivery in preclinical phases highlight them as promising alternatives to conventional nanomaterials, as they benefit from the inherent favorable properties of DNA including biocompatibility and precise spatial addressability. By incorporating targeting aptamers and responsive properties into the nanocarrier design, more selective DNA origami-based nanocarriers are successfully prepared. On the other hand, current systems remain poorly understood in terms of biodistribution, final fate, and controlled drug release. As such, advances are needed to translate this material platform in its full potential for therapeutic applications.

2021

Cu/Metal Oxide Hybrid Nanocrystals as Electrocatalysts for the CO2 Reduction Reaction

Seyedehbehnaz Varandili

The electrochemical CO2 reduction reaction (CO2RR) is envisioned to play a significant role in achieving carbon neutrality while contributing to storing renewable energies. Cu-based materials are among the most promising electrocatalysts. However, 16 different products are obtained from a Cu foil, therefore strategies to make more selective catalysts are needed. This thesis explores Cu/metal oxide interfaces as a mean to direct selectivity towards hydrocarbon products in CO2RR. The aim is to deviate from the scaling relationships which exist on single metal surfaces. Cu/metal oxides hybrid nanocrystals (HNCs) with tunable morphologies and spatial configuration are synthesized via colloidal chemistry. The achieved level of control and tunability is demonstrated to be crucial in understanding the impact of these parameters on the reaction outcome. First, we start by designing a synthesis procedure for Cu/CeO2-x heterodimers (HDs) and then we study their catalytic behavior toward CO2RR. We show that the Cu/CeO2-x interface promotes CO2RR, particularly methane with around 54% selectivity, which is ~5 times higher than those obtained with a physical mixture of isolated Cu and CeO2-x nanocrystals, thus highlighting the important role played by the intimate bonding at the interface. A miscellanea of characterization techniques indicates the higher extent of ceria reduction in the HDs during CO2RR. DFT elucidates the presence of unique catalytic motifs that enable bidentate adsorption of critical intermediates at both Cu and the CeO2-xO-vacancy site, creating an opportunity for reaction engineering beyond scaling relationship limitations. Having assessed the importance of the Cu/ceria interface, we investigate the impact of increasing the Cu/CeO2 interfacial area on the CO2RR outcome. We find that increasing the number of ceria domain around copper inhibits CO2RR. This result highlights that a balance between interfacial area and accessibility to the active catalytic sites must be sought after to exploit synergies arising at the interface between different domains. Finally, we explore Cu/ZnO HNCs as CO2RR pre-catalysts to elucidate the structural and compositional parameters which direct the reaction pathway towards ethanol. Using different sizes of ZnO seeds for the synthesis of Cu/ZnO HNCs, we are able to achieve two different configurations including ZnO decorated Cu core (Cu@ZnO) and Cu decorated ZnO core (ZnO@Cu) HNCs. Upon activation via cathodic voltage, we find that Cu@ZnO HNCs favor methane production while ZnO@Cu exhibits a noticeable selectivity toward ethanol. Operando XAS, XPS, and electron microscopy reveal that both HNCs transform into Cu@CuZn core@shell NCs during the activation step. However, the Zn content in the surface alloy (i.e. degree of alloying) and the presence of surface cationic Zn species are found to be crucial in determining the selectivity of these catalysts, which possibly elucidates some of the contrasting results reported on Cu-Zn across the literature. Overall, this thesis showcases the importance of well-defined and tunable HNCs as platforms to study structure/properties relations and to advance the current knowledge in CO2RR. Furthermore, the library of nanomaterials accessible through the developed synthesis routes might also be utilized in the future to investigate other catalytic reactions and applications where the hybrid interfaces might be relevant.

EPFL2021

Investigating the molecular basis of ligand binding to and activation of olfactory receptors in living cells

Paulina Izewska

The olfactory system is a highly specialized chemodetector capable of discriminating between thousands of volatile chemical substances. This enormous capacity of chemical recognition is based on a large family of olfactory receptors (ORs). Despite the fact that quite a number of putative ORs have been cloned, only limited progress has been made in functional expression of these receptors in heterologous cells. In order to functionally express odorant receptors we have established a suitable expression system in HEK 293 cells. The co-expression of the Gαq protein together with odorant receptors allowed the study of OR activation by calcium imaging. To analyze the structural properties of odorant molecules essential for OR5 and hOR17-4 binding and activation, we developed an efficient functional assay based on optical read-out of odorant responses by intracellular calcium imaging on living cells. Investigation of the ligand specificity of the two ORs derived from different species, but exhibiting significant homology on amino acid sequence level, showed a high degree of similarity in their responsive odorant molecule spectrum. A comparison of the molecular properties of odorant compounds crucial for OR5 and hOR17-4 binding and activation revealed a classification in two different structural groups. The first one comprises aromatic hydrocarbons with variable structures and sizes and the second group consists of aliphatic odorant molecules which are mainly aldehydes with variable carbon chain lengths. To elucidate further the function of ORs, the computational modeling to predict the binding site of OR5 and the docking of the small, hydrophobic odorant molecule, safrole were determined. Our findings show that the safrole binding site in OR5 involves the helical domains TM3, TM5 and TM6, which is consistent with earlier computational studies on rat I7 and mouse OR-EG receptors. The key amino acid residues appear to be hydrophobic and the van der Waals forces are implicated in safrole binding to the OR5 receptor. The mutations of the amino acid residues predicted by safrole docking analyses, Val202, Phe251 and Leu258 to Ala, indicate that these residues play an important role in OR5 activation.