Erythrocyte Membrane Incorporation Modulates the Stability and Fusion-Associated Behavior of Biomimetic Liposomes

Abstract

This study investigates how membrane composition governs the stability and fusogenic behavior of biomimetic hybrid liposomes composed of phosphatidylcholine (PC) and red blood cell (RBC) membrane fragments. While prior work on RBC-derived hybrid systems has largely focused on biological performance metrics such as circulation time, cellular uptake, and immune evasion, the biophysical and thermodynamic consequences of membrane hybridization at the molecular level remain poorly explored. Here, we address this gap through a systematic, multitechnique physicochemical characterization of a series of RBC-hybrid liposome formulations spanning a range of lipid:protein ratios. A comprehensive physicochemical characterization was performed using electron spin resonance (ESR) to probe membrane dynamics, microdifferential scanning calorimetry (microDSC) to assess thermotropic behavior, and isothermal titration calorimetry (ITC) to examine membrane energetics, solubilization, and intervesicular interactions. Incorporation of RBC membrane fragments increased membrane rigidity, as indicated by ESR and microDSC, and was associated with enhanced resistance to Triton X-100-induced solubilization, a finding attributed to compositional heterogeneity and protein–lipid organization introduced by membrane hybridization. ITC-based interaction assays further showed that cationic hybrid liposomes exhibited stronger fusion-associated signatures than their conventional counterparts, indicating that RBC membrane incorporation significantly alters interfacial membrane behavior in ways that cannot be explained by lipid composition alone. Together, these results show that RBC membrane content modulates both the structural stability and interaction profile of liposomes, establishing a biophysical framework for understanding how biomembrane-derived components reshape vesicle behavior and providing insights and useful design parameters for the development of robust biomimetic lipid-based delivery systems.