(W1130-03-17) Molecular Dynamics Insights into Cryoprotectant-Mediated Stabilization of Amphotericin Liposomes During Freeze-Drying: A Comparative MD and SA-MD Study

Purpose: This study investigates the comparative effects of three common cryoprotectants, sucrose, trehalose, and mannitol on the structural stability of lipid bilayers during molecular dynamics (MD) and simulated annealing MD (SA-MD) simulations of Amphotericin liposome. Emphasis is placed on analyzing the radius of gyration (Rg), root-mean-square deviation (RMSD), hydrogen bonding, and total system energy to evaluate the degree of interaction and stabilization each excipient offers to the lipid headgroups. Cryoprotectants play a crucial role in preserving membrane integrity during thermal stress and freeze thaw cycles by replacing water molecules and forming hydrogen bonds with lipid headgroups. The goal of this work is to establish a comparative study of these molecules based on their stabilizing capacity of liposome during freeze drying. Such insights are essential for optimizing cryoprotective formulations in pharmaceuticals research involving membrane models.

Methods: All-atom structures of hydrogenated soy phosphatidylcholine (HSPC), 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG), cholesterol, amphotericin B, sucrose, trehalose, and mannitol were downloaded in SDF format from publicly available chemical structure databases. These compounds were prepared using LigPrep (Schrödinger Materials Science Suite 2024-5) to generate energy-minimized, three-dimensional structures with proper protonation states and stereochemistry for subsequent molecular modeling and simulation. The simulation system comprised a total of lipid molecules, water molecules, and Amphotericin B molecules, arranged within a periodic simulation box to model the membrane–drug–solvent interactions under cryoprotective conditions. Simulated annealing was performed over a 10 ns trajectory, during which the system temperature was gradually reduced from 300 K to 233 K to mimic a controlled cooling process. The annealing protocol was applied to assess thermally induced structural changes and cryoprotectant interactions under near-freezing conditions. The simulation time for each cryoprotectant was 100 nanosecond (ns) with temperature 300 K for conventional MD and 233 K for Simulated annealing-MD with NPT ensemble class.

Results: The cryoprotective efficacy of sucrose, trehalose, and mannitol in preserving the structural integrity of Amphotericin liposomes under freeze-drying conditions was systematically assessed through an in-depth molecular dynamics investigation employing both conventional Molecular Dynamics (MD) and Simulated Annealing Molecular Dynamics (SA-MD) simulations. Key structural and thermodynamic descriptors including Root Mean Square Deviation (RMSD), Radius of Gyration (Rg), hydrogen bonding interactions, and total system energy were analyzed to elucidate the stabilizing mechanisms conferred by each cryoprotectant. RMSD analysis revealed that both sucrose and trehalose effectively minimized lipid fluctuations under SA-MD conditions, with RMSD values reduced to 45–60 Å, compared to 50–65 Å in MD simulations, indicating enhanced membrane rigidity. Mannitol, however, showed negligible variation between MD and SA-MD, maintaining an RMSD range of 50–65 Å, thereby signifying limited stabilizing capacity. Similarly, Rg analysis demonstrated that sucrose achieved the most compact lipid headgroup packing under SA-MD (44.5–45.5 Å), closely followed by trehalose (44.5–46.0 Å), whereas mannitol exhibited a broader Rg range (45.0–47.5 Å), indicative of membrane loosening and insufficient structural compaction. Hydrogen bonding analysis further supported these findings, with sucrose forming the highest number of interactions with lipid molecules (~8000 in MD, increasing to ~8500 in SA-MD), followed by trehalose (~7900–8400), while mannitol exhibited the fewest interactions, plateauing below ~8100 in both simulations. These trends highlight the superior water replacement and headgroup stabilization capabilities of sucrose and trehalose compared to mannitol. The total energy profiles also reflected this hierarchy: sucrose exhibited the most thermodynamically favorable stabilization under SA-MD (310,500–311,000 kcal/mol), with a marked decrease from its MD energy (~320,000–322,000 kcal/mol), while trehalose stabilized near ~308,000–310,000 kcal/mol in both conditions, suggesting moderate energy minimization. Conversely, mannitol, despite reaching a low MD energy (~295,000–298,000 kcal/mol), did not exhibit energy reduction under SA-MD, remaining around ~310,000 kcal/mol, indicating inconsistent thermodynamic behavior and poor stabilization efficiency. Collectively, these results demonstrate that sucrose and trehalose offer comparable and superior cryoprotective effects, while mannitol shows limited ability to preserve the structural and energetic integrity of Amphotericin liposomes under freeze-drying stress

Conclusion: The molecular dynamics evaluation demonstrates that both sucrose and trehalose provide superior structural and thermodynamic stability to Amphotericin liposomes during freeze-drying, particularly under simulated annealing conditions. Their ability to enhance headgroup packing, form extensive hydrogen bonds, and maintain lower system energy in SA-MD reinforces their roles as effective cryoprotectants. In contrast, mannitol shows limited capability and may contribute to destabilization of the lipid bilayer under stress. These findings underscore the critical need for cryoprotectant optimization in liposomal formulation design for enhanced stability and performance during lyophilization.

References: Ermilova I, Lyubartsev A, Kocherbitov V. Sucrose versus Trehalose: Observations from Comparative Study Using Molecular Dynamics Simulations. ACS Omega. 2024


Figure 1: Trajectory analysis of simulated systems containing MD and SA-MD


Figure 2: Snapshots of trajectory of simulated system containing cryoprotectants and lipids


Table 1: MD and SA-MD trajectory analysis results