A Systematic Investigation of Cellular Interactions with Lipid-Based Lyotropic Liquid Crystalline Nanoparticles

<p dir="ltr">The clinical implementation of lipid nanoparticles (LNPs) in the first successful nucleic acid-based vaccines marks a milestone in drug delivery, demonstrating the transformative potential of these nanocarriers in life-changing therapeutics. A major challenge in drug delivery lies in overcoming biological and transport barriers that limit the efficacy of therapeutics. For example, many drugs fail to surpass biological barriers such as the plasma membrane or the blood-brain barrier. Furthermore, hydrophobic drugs suffer from poor water solubility, reducing their bioavailability in the bloodstream. Similarly, fragile cargoes like nucleic acids are rapidly degraded by enzymatic activity in the body before reaching their intended targets. Even when bioactive molecules reach their cellular targets and undergo internalization, intracellular barriers present a further obstacle to their therapeutic efficacy. Endosomal entrapment, lysosomal degradation, and cellular recycling pathways often prevent therapeutics from reaching their subcellular targets. These challenges significantly reduce the efficiency of drug delivery systems, necessitating novel strategies to improve intracellular cargo transport.</p><p dir="ltr">LNPs have emerged as a promising solution due to their ability to encapsulate hydrophobic, hydrophilic, and amphiphilic compounds, effectively improving drug solubility, stability, and cellular uptake. By facilitating the transport of bioactive cargoes across the plasma membrane, LNPs function as a "Trojan horse" for intracellular delivery. Despite this advancement, endosomal entrapment and degradation remain a major limitation to their therapeutic efficacy. Studies estimate that less than 5% of nucleic acids successfully escape the endosome post-LNP endocytosis, highlighting the need for improved delivery strategies to enhance therapeutic outcomes.</p><p dir="ltr">Non-lamellar lyotropic liquid crystalline nanoparticles (LLCNPs), including cubosomes, hexosomes, and micellar cubosomes, have emerged as a promising subclass of LNPs due to their highly ordered internal nanostructures and unique physicochemical properties. These nanoparticles exhibit high surface-to-volume ratios for efficient drug encapsulation, biocompatibility, sustained drug release properties, and importantly, the ability to fuse with cellular membranes, offering a potential strategy to circumvent endosomal entrapment and degradation. Despite extensive research on their design and physicochemical properties, the biological interactions of LLCNPs that govern intracellular delivery—particularly how their nanostructures affect cellular uptake, internalization pathways, intracellular fate, endosomal escape, and membrane fusion—remain largely unexplored. Understanding these interactions is crucial for elucidating their mechanism of action and their subsequent therapeutic efficacy.</p><p dir="ltr">This thesis systematically investigates how LLCNP nanostructure influences cellular uptake, internalization mechanisms, and intracellular fate, providing critical insights into their potential as effective nanocarriers for drug delivery. Chapter 3 explores the cellular uptake efficiencies of LLCNPs with varying internal nanostructures (liposomes, cubosomes, hexosomes, and micellar cubosomes) in epithelial cells, using flow cytometry and confocal microscopy. This study revealed that non-lamellar LLCNPs exhibit significantly higher and more sustained cellular interactions than conventional liposomes, which are widely used in clinical applications. By optimizing LLCNP formulations to control size, surface charge, and surface stabilizer, this study isolated the effect of nanostructure on cellular interactions, highlighting non-lamellar structures as particularly effective for their enhanced cellular uptake.</p><p dir="ltr">Chapter 4 investigated the internalization pathways governing these interactions, using pharmacological inhibitors and endocytic markers (analyzed via flow cytometry and confocal microscopy). This study revealed that liposomes primarily rely on endocytic pathways, whereas non-lamellar LLCNPs relied heavily on a passive non-endocytic mechanism for their internalization, likely membrane fusion. Interestingly, macropinocytosis emerged as the dominant endocytic pathway for non-lamellar LLCNPs, suggesting their potential for targeting immune cells and Ras-activated cancer cells, where macropinocytosis is highly active. Furthermore, the ability of non-lamellar LLCNPs to bypass endocytic pathways highlights their potential for therapeutic delivery in diseases characterized by impaired endocytosis, such as Type A Niemann-Pick disease and Alzheimer’s disease, and their potential in circumventing the endosomal pathways altogether. Given these findings and the enhanced cellular interactions observed in Chapter 3, it is likely that membrane fusion plays a critical role in the uptake of non-lamellar LLCNPs, particularly cubosomes, which exhibited the highest levels of cellular uptake. </p><p dir="ltr">Despite the potential of LNP-mediated membrane fusion to circumvent endosomal entrapment and enable direct cytosolic delivery, this dynamic process remains underexplored, with previous studies limited to model membranes or inferred from FRET-based fusion assays. Direct visualization of this phenomenon in mammalian cells has remained elusive due to the nanoscale dimensions of LNPs. Chapter 5 addresses this critical gap by using a suite of advanced microscopy techniques, including electron microscopy (TEM, SEM, Cryo-SEM), fluorescence-based assays, and live cell fluorescence imaging. This study provides the first direct evidence of cubosome-mediated membrane fusion with mammalian plasma and endosomal membranes, demonstrating their ability to circumvent endosomal degradation and deliver cargo directly into the cytosol. Cubosomes were selected as a model system due to their demonstrated fusogenic properties, their enhanced cellular uptake, and their preferential utilization of non-endocytic pathways observed in Chapters 3 and 4. This study also highlighted the unique fusogenic properties of cubosomes driven by their physical properties and nanostructure, distinguishing them from other LNPs.</p><p dir="ltr">To refine the nanoscale imaging of cubosome-membrane fusion, Chapter 6 develops an optimized Cryo-Electron Tomography (Cryo-ET) workflow, designed to enable high-resolution 3D imaging of membrane fusion events in a frozen-hydrated state. A major barrier to successful Cryo-ET imaging lies in sample preparation, as conventional workflows require highly complex techniques such as cryogenic fluorescence light microscopy (cryo-fLM) and cryogenic focused ion beam (cryo-FIB) milling, limiting accessibility for directly visualizing key biological processes such as membrane fusion. This chapter presents an optimized and accessible cryo-ET workflow, incorporating pre-optimized LNP-cell conditions, ultrathin cell lines (negating the need for cryo-FIB), fixation protocols, and fluorescence-based targeting for region selection (negating the need for cryo-fLM). This methodology streamlines Cryo-ET sample preparation, making the high-resolution imaging of LNP-membrane fusion more feasible, and providing a powerful tool for future investigations into the spatio-temporal dynamics of LNP-mediated fusion and intracellular delivery.</p><p dir="ltr">Collectively, this thesis advances our understanding of LLCNP cellular interactions, internalization mechanisms, and intracellular fate, establishing nanostructure as a key design principle for enhanced intracellular drug delivery. The presented findings provide a framework for optimizing LLCNP formulations for targeted applications, such as the delivery of nucleic acids for cell transfection (leveraging cubosomes’ membrane fusion capabilities) or cancer therapeutics utilizing macropinocytosis-mediated uptake. By bridging fundamental biophysical insights with translational applications, this research lays the groundwork for next-generation LLCNP-based drug delivery technologies that harness nanostructure-driven intracellular delivery mechanisms.</p>