Mapping Berberine distribution in liposomes: the role of drug-phospholipid interactions in localization and release

Liposomes are versatile nanocarriers capable of encapsulating drugs within distinct compartments. However, the impact of drug localization on release kinetics remains poorly understood, particularly for amphiphilic compounds. In this study, Berberine was employed as a model amphiphilic drug to investigate how drug-lipid interactions and membrane composition influence its intra-liposomal distribution and release behavior. Spectroscopic and calorimetric analyses revealed that Berberine predominantly localizes within the aqueous core of liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), while the incorporation of 1,2- dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) promotes additional partitioning into the lipid bilayer and adsorption onto the liposome surface. To systematically study these effects, liposomal formulations containing increasing DPPG content (0 %, 10 %, 30 %) were prepared using dual centrifugation. Molecular dynamics simulations complemented experimental findings, demonstrating that higher DPPG levels enhance electrostatic interactions with Berberine and induce interdigitated bilayer structures characterized by looser lateral packing, increased area per lipid, and reduced bilayer thickness. In vitro release studies showed that liposomes with a lower content of DPPG exhibited a slow, sustained biphasic release profile, while liposomes with a higher content of DPPG released Berberine rapidly, consistent with dominant surface localization. This faster release is likely due to side of the membrane Berberine localizes during loading rather than accelerated membrane permeation. Reduced drug loading favored partitioning of a greater fraction of drug to the inner side of the bilayer, thereby attenuating release rates. Together, these results highlight the central role of drug localization, governed by loading procedure, electrostatic interactions and membrane architecture, in dictating liposomal release kinetics. This work provides mechanistic insight to inform the rational design of liposomal delivery systems for amphiphilic and cationic compounds with attributes such as Berberine.