Elucidating Structural Configuration of Lipid Assemblies for mRNA Delivery Systems.

The development of mRNA delivery systems utilizing lipid-based assemblies holds immense potential for precise control of gene expression and targeted therapeutic interventions. Despite advancements in lipid-based gene delivery systems, a critical knowledge gap remains in understanding how the biophysical characteristics of lipid assemblies and mRNA complexes influence these systems. Herein, we investigate the biophysical properties of cationic liposomes and their role in shaping mRNA lipoplexes by comparing various fabrication methods. Notably, an innovative fabrication technique called the liposome under cryo-assembly (LUCA) cycle, involving a precisely controlled freeze-thaw-vortex process, produces distinctive onion-like concentric multilamellar structures in cationic DOTAP/DOPE liposomes, in contrast to a conventional extrusion method that yields unilamellar liposomes. The inclusion of short-chain DHPC lipids further modulates the structure of cationic liposomes, transforming them from multilamellar to unilamellar structures during the LUCA cycle. Furthermore, the biophysical and biological evaluations of mRNA lipoplexes unveil that the optimal N/P charge ratio in the lipoplex can vary depending on the structure of initial cationic liposomes. Cryo-EM structural analysis demonstrates that multilamellar cationic liposomes induce two distinct interlamellar spacings in cationic lipoplexes, emphasizing the significant impact of the liposome structures on the final structure of mRNA lipoplexes. Taken together, our results provide an intriguing insight into the relationship between lipid assembly structures and the biophysical characteristics of the resulting lipoplexes. These relationships may open the door for advancing lipid-based mRNA delivery systems through more streamlined manufacturing processes.

Characterizing the Supported Lipid Membrane Formation from Cholesterol-Rich Bicelles.

Supported lipid bilayers (SLBs) are simplified model membrane systems that mimic the fundamental properties of biological cell membranes and allow the surface-sensitive tools to be used in numerous sensing applications. SLBs can be prepared by various methods including vesicle fusion, solvent-assisted lipid bilayer (SALB), and bicelle adsorption and are generally composed of phospholipids. Incorporating other biologically relevant molecules, such as cholesterol (Chol), into SLBs has been reported with the vesicle fusion and SALB methods, whereas it remains unexplored with the bicelle absorption method. Herein, using the quartz crystal microbalance-dissipation (QCM-D) and fluorescence microscopy techniques, we explored the possibility of forming SLBs from Chol-containing bicelles and discovered that Chol-enriched SLBs can be fabricated with bicelles. We also compared the Chol-enriched SLB formation of the bicelle method to that of vesicle fusion and SALB and discussed how the differences in lipid assembly properties can cause the differences in the adsorption kinetics and final results of SLB formation. Collectively, our findings demonstrate that the vesicle fusion method is least favorable for forming Chol-enriched SLBs, whereas the SALB and bicelle methods are more favorable, highlighting the need to consider the application requirements when choosing a suitable method for the formation of Chol-enriched SLBs.