UC Davis

Silicon is the second most abundant element in Earth’s crust and has no known element–specific toxicity. Despite being carbon’s elemental “cousin”, the silicon atom contains unique properties that can be harnessed in medicinal chemistry and chemical biology to improve chemical and biological properties. Some of these unique properties that are applicable to chemical biology are silicon’s enhanced lipophilic character and larger size compared to carbon. Silicon’s larger atomic radius results in 20% longer bond lengths compared to regular carbon–carbon bonds. These longer bond lengths in silicon–carbon bonds increase the surface area for Van der Waals intermolecular forces, allowing for greater hydrophobic interactions. Modification of hydrophobic interactions are particularly relevant in (i) medicinal chemistry for enhancing small molecule binding in hydrophobic binding pockets of proteins and increasing permeability of lipophilic and amphiphilic molecules across cell membranes, and (ii) chemical biology for modulating membrane fluidity and stability related to lipid nanoparticles (LNPs). Chapter 1 summarizes the relevant properties of silicon in medicinal chemistry and chemical biology. A few specific examples are given that showcase how incorporation of silicon into small molecules increased their therapeutic properties such as potency and stability. Hydrosilylation is also described as a key synthetic strategy to access modular silicon–containing molecules. Chapter 1 also summarizes the use of LNPs and the history of amphiphilic lipids that have been synthesized with varying polar head groups and nonpolar tails to optimize LNP properties and nucleic acid delivery. LNPs have widely been used for nucleic acid delivery and are composed of amphiphilic lipids that contain a polar head group and nonpolar lipid tails. There are five classes of lipids used in LNPs. The cationic lipid component has a positively charged polar head group that facilitates in encapsulation of negatively charged RNA and DNA and cellular uptake. The polar head group of ionizable lipids is pH dependent, with the head group becoming protonated in the endosome under acidic conditions, facilitating in endosomal release of nucleic acid inside the cell. Zwitterionic phospholipids and cholesterol are essential for controlling membrane fluidity and structural stability of the LNP. Lastly, PEGylated lipids increase in vivo stability, biodistribution and circulation time of LNPs. Several synthetic modifications have been made to the polar head group of amphiphilic lipids to control and improve LNP properties and nucleic acid delivery. However fewer examples of nonpolar tail modifications exist, with the most common examples including saturated tails with varying lengths, unsaturation, and branching. Chapters 2 and 3 present my work for the synthesis and biophysical and biological evaluation of cationic and ionizable silyl lipids for their use in lipid nanoparticles and mRNA delivery. Lipid nanoparticles were formulated with ionizable and cationic silyl lipids and the following properties were evaluated: (i) size using dynamic light scattering (DLS) and cryo–TEM, (ii) encapsulation efficiency using the RiboGreen fluorescence assay, (iii) membrane fluidity using the C-Laurdan fluorescence assay and (iv) mRNA transfection efficiency in HEK293T cells. Chapter 2 demonstrates the synthesis and evaluation of cationic silyl lipids with modular tail structures to control particle properties, with key findings including (i) shorter silyl lipid tails (many containing phenyl rings) generally form particles with enhanced RNA encapsulation efficiency and mRNA transfection efficiency in HEK293T cells, (ii) the silyldimethyl group in the lipid tail enhances particle stability compared to a methylene and (iii) varying the silyl lipid tail structure enables control of membrane fluidity, with the silyldimethyl group imparting enhanced membrane fluidity compared to cis alkene and methylene–containing control lipids. Chapter 3 builds off this work by demonstrating the synthesis and evaluation of ionizable silyl lipids for mRNA delivery. Cryo–TEM, RiboGreen and C-laurdan assay, and cell transfection data supports that modifying the silyl lipid tail structure and the number of silyl lipid tails (i.e. single vs double chain lipids) can be leveraged to control particle properties, with particles composed of single chain lipids demonstrating distinct membrane fluidity and mRNA delivery profiles compared to double chain lipids. Appendix A discusses the evaluation of DNA encapsulation efficiency, encapsulation rate, and transfection efficiency for the cationic and ionizable silyl lipids. Appendix B contains all relevant NMR data.