Physicochemical Characterization of Anionic Lipid Membranes under Low and Physiological Ionic Strength: Effects of Moxifloxacin Assessed by Calorimetry, Spin-Label, Steady-State, and Time-Resolved Fluorescence.

In the present study, we comparatively investigate the interaction between the antibiotic moxifloxacin (MFX) and extruded (100 nm) lipid vesicles composed of the anionic lipid 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), under both low ([NaCl] = 3 mmol L-1) and physiological ionic strength conditions ([NaCl] = 150 mmol L-1). By employing differential scanning calorimetry (DSC) and electron spin resonance (ESR) spectroscopy with spin-labeled lipids positioned at two distinct depths within the bilayer, we evaluated structural alterations induced by increasing concentrations of MFX. Additionally, taking advantage of the intrinsic fluorescence of the antibiotic, we monitored structural changes and determined its affinity for the anionic bilayers upon exposure to rising concentrations of liposomes under different configurations: gel and fluid phases. MFX was found to induce a narrowing of the main gel-to-fluid phase transition peak of DPPG under both ionic strengths, indicating enhanced cooperativity of the thermal lipid transition. ESR spectra of spin labels inserted at the fifth carbon position, near the interfacial region between the polar headgroups and the hydrophobic core, and deeper within the bilayer revealed that MFX promotes tighter lipid packing and decreases the local polarity of the membrane interior, independently of salt concentration. Upon increasing the concentration of anionic liposomes, the MFX emission displays a relative redshift. The data suggest that, upon interaction with the anionic bilayers, a fraction of the zwitterionic MFX molecules undergoes protonation, presumably driven by a locally low pH microenvironment at the vesicle interface. MFX fluorescence lifetimes are enhanced by its association with gel and fluid liposomes, enabling the determination of membrane-water partition coefficients, which were found to be K p = (2.5 ± 0.2) × 104 and (1.4 ± 0.1) × 104 for the gel and fluid phases, respectively, under low ionic strength conditions. Furthermore, given the relevance of antibiotic-membrane interactions to antimicrobial efficacy, toxicity, and drug delivery strategies, the findings reported herein advance the understanding of the physicochemical behavior of MFX in lipid environments and may guide the rational development of membrane-targeting antibiotics and lipid-based delivery systems.