Liposome−Prilocaine Interaction Mapping Evaluated through STD NMR and Molecular Dynamics Simulations

Abstract

We have examined the interaction of the neutral and protonated species of the local anesthetic prilocaine (PLC) with phosphatidylcholine (PC) bilayers combining experimental ((1)H NMR) and theoretical (molecular dynamics simulations) approaches. DOSY experiments allowed the determination of the association constants of protonated (Ka = 9 L/mol) and neutral (Ka = 21 L/mol) PLC to egg PC liposomes. Saturation transfer difference (STD) experiments showed a different trend depending on pH: At high pH the PLC hydrogen saturation was essentially uniform and at pH 5.5 the experiments show an enhancement of the aromatic moiety hydrogen saturation, with respect to the tail. Molecular dynamics simulations, performed with PLC molecules on planar bilayers of palmitoyloleyl-PC, revealed a preferential orientation for the protonated PLC species at the polar interface of the bilayer, and a nonoriented and deeper insertion for neutral PLC. Such preferential location of protonated and neutral PLC inside the bilayer can be described as different transient sites which could modulate the access of these molecules to their binding site(s) in the voltage-gated sodium channel, justifying differences in the anesthetic's potency upon ionization.

Prilocaine–cyclodextrin–liposome: effect of pH variations on the encapsulation and topology of a ternary complex using 1H NMR

“…Investigating the topologies of PLC in β‐CD in EPC liposomes at different pHs by NMR required full characterization of simple binary mixtures in aqueous solutions of PLC, β‐CD and EPC. The pH influence on PLC/EPC complex topology was fully assessed by applying STD, and molecular dynamics simulations, revealing that at pH 10 the PLC molecules were mostly imbedded into the liposome, whereas at pH 5.5, the PLC behavior was modulated by the dynamic equilibrium between the charged (in the aqueous phase) and uncharged species (in the liposome)26 (Fig. 1).…”

“…3) indicated an interaction between PLC and the liposome vesicles, causing saturation transference from the liposomes to all PLC hydrogens and indicating that PLC is imbedded into the liposome vesicles. The EPC saturation transfer was more efficient for the PLC aromatic moiety (100%) which might indicate a longer residence time in the liposome vesicles, consistent with previously published results using PLC‐EPC 26…”

“…Finally, at pH 5.5 there is again an almost homogeneous saturation transfer to all hydrogens but the effect is really weak (about 0.45), indicating a lower apparent association constant and that most molecules are out of the liposome. Hydrogens H‐10 and H‐13 were not taken into account because at lower pH they overlapped, preventing any detailed analysis, as previously reported 26…”

NMR Spectroscopy for Studying Interactions of Bioactive Molecules

“…The results pointed towards the fact that improved solubility in biological media could be achieved by choosing a six-atom side chain length and that enhancement of membrane interactions/penetration could be achieved by hydroxyl substitutions on the C6 lateral chain. Cabeça et al [108] applied molecular dynamics (MD) simulations and NMR spectroscopy to examine the interaction of the neutral and protonated species of the local anaesthetic prilocaine (PLC) with phosphatidylcholine (PC) bilayers. The diffusion-ordered 2D NMR spectroscopy (DOSY) experiments showed that neutral PLC has a higher affinity for the liposome environment.…”

CHARMM‐based parameterization of neutral articaine—A widely used local anesthetic

“…From a theoretical perspective, atomistic molecular dynamics (MD) computer simulations are being successfully applied to investigate the effects of various LAs on model phospholipidic membranes at a molecular level18–23. However, because of the wide variety of these compounds, it is often needed to develop new interaction potential parameters that allow the study of these systems by MD simulations.…”