(197a) Fusogenic Liposome Formation: A Coarse-Grained Molecular Dynamics Simulations

Liposomes are vesicles composed of a lipid bilayer and/or a concentric series of multiple bilayers commonly used as carriers for drug delivery purposes. In contrast to the conventional pathway of cell uptake, previous studies have proposed that fusogenic liposomes have the advantages of fusing with the cell membrane and directly releasing the cargo to the cytosol. However, there is a lack of deep understanding of the structural impact of the individual lipid on its assemblies, and uncovering the driving force that differentiates the route of the cellular uptake has been challenging. Here, we implemented coarse-grained molecular dynamics (CG-MD) simulations to predict the physicochemical properties of new lipids, simulate their propensity to self-assemble into liposomes, and determine the correlation between liposome size and lipid structural properties.

Methods:

The CG-MD simulations were implemented using the GROMACS software package with the MARTINI 3.0 force fields, and the trajectory analyses were conducted using MDAnalysis and PackMem. The all-atom model of examined new lipids, which composed of an amino acid-based head group, a spacer group, and two alkyl tail groups of various lengths, was first constructed using the webserver LigParGen. The new lipid model was then mapped and parameterized into the coarse-grained model. The constructed new CG lipid model was energy-minimized with the steepest descent algorithm, followed by a 10 ns equilibration in an isothermal−isochoric ensemble at ambient temperature. The cubic simulation system of various sizes (10 nm³, 25 nm³, 50 nm³, and 100 nm³) was built as mixture of water and randomly distributed lipids. The 200-ns simulation production runs were performed in an isothermal-isobaric ensemble using the Nosé−Hoover thermostat and the Parrinello−Rahman barostat at a pressure of 1 bar. The simulations were carried out using periodic boundary conditions in a 20-fs time step and the final trajectory was extracted for further physicochemical property analysis. All computations were performed using the High-Performance Computing (HPC) Supercomputer Cluster at Genentech.

Results:

The CG-MD simulations indicate that the examined lipids self-assemble into liposomes and micelles of various shapes and sizes within 100ns into the production run. Analysis of the radius of gyration showed that all liposomes formed were initially unstable and experienced significant fluctuations in their morphology. During this early stage, the liposomes usually transitioned from an elongated shape to an elliptical shape, eventually stabilizing into a mostly spherical shape that persisted for the duration of the simulation. Furthermore, the simulations showed that the thickness of fusogenic liposome bilayer was influenced by only certain types of amino acid head group, while the length of the spacer and the alkyl tail groups influence the liposome bilayer thickness independently from the head group. The curvature analysis provided detailed insights into the behavior of the membrane bilayer, revealing that the inner monolayer was more curved than the outer monolayer. The evaluation of three-dimensional packing defects in the membrane bilayer showed that liposome formation in this study was characterized by more deep defects than shallow defects, which indicates the regions where the hydrophobic lipid tails are transiently exposed to the aqueous polar environment leading to unstable liposomal structure. The simulation findings will be further validated through experimental studies using the same fusogenic lipids.

Conclusions:

While the simulation exercises did not accurately replicate the experimental particle size, potentially due to computational limitations, they still effectively uncovered the mechanism behind the formation of liposomes and their structural properties using newly designed fusogenic lipids with varying head groups, spacer lengths, and tail lengths. The MD computational platform developed in this study has proven to be a powerful tool that can help to facilitate the synthesis and testing of fusogenic lipids with specific desired properties. Not only this, but it is also capable of guiding the interpretation, explanation, and direction of experimental studies in this area.

Keywords:

Fusogenic liposomes, Molecular dynamics simulations, Coarse-grained, Membrane defects, Membrane curvature.

Figure 1. CG-MD fusogenic liposome self-assembly (left) and cross-sectional view (right).