Nuclear Magnetic Resonance Spectroscopy: A Multifaceted Toolbox to Probe Structure, Dynamics, Interactions, and Real-Time In Situ Release Kinetics in Peptide-Liposome Formulations

Abstract

Liposomal formulations represent attractive biocompatible and tunable drug delivery systems for peptide drugs. Among the tools to analyze their physicochemical properties, nuclear magnetic resonance (NMR) spectroscopy, despite being an obligatory technique to characterize molecular structure and dynamics in chemistry as well as in structural biology, yet appears to be rather sparsely used to study drug-liposome formulations. In this work, we exploited several facets of liquid-state NMR spectroscopy to characterize liposomal delivery systems for the apelin-derived K14P peptide and K14P modified by Nα-fatty acylation. Various liposome compositions and preparation modes were analyzed. Using NMR, in combination with cryo-electron microscopy and dynamic light scattering, we determined structural, dynamic, and self-association properties of these peptides in solution and probed their interactions with liposomes. Using 31P and 1H NMR, we characterized membrane fluidity and thermotropic phase transitions in empty and loaded liposomes. Based on diffusion and 1H NMR experiments, we localized and quantified peptides with respect to the interior/exterior of liposomes and changes over time and upon thermal treatments. Finally, we assessed the release kinetics of several solutes and compared various formulations. Taken together, this work shows that NMR has the potential to assist the design of peptide/liposome systems and more generally drug delivery systems.

Analytical Techniques for the Characterization and Quantification of Monoclonal Antibodies

“…Recently, investigations of both the protein and stability of mAb formulations have been performed using 1D 1 H NMR signal intensity through the protein degradation i.e., aggregation or fragmentation [ 72 ]. Furthermore, the combination of liquid-state NMR spectroscopy with cryo-electron microscopy and dynamic light scattering reported to play a critical role in drug delivery systems through characterizing various aspects of liposome-peptide formulation, such as peptide and liposome assembly, stability of the formulation, their mutual interactions, and the peptide release profiling [ 73 ].…”

Liposomes: preparation and characterization with a special focus on the application of capillary electrophoresis

“…To ensure the proper function of liposomes as carriers for bioactive substances, it is necessary to characterize their properties, such as size, lamellarity, surface charge, quantitative composition, and encapsulation efficiency [ 31 ]. Commonly used methods to analyze and characterize liposomes include 31 P nuclear magnetic resonance (NMR) [ 32 ] which is used for studying the membrane fluidity and thermotropic phase transitions, dynamic light scattering (DLS) [ 33 ], atomic force microscopy (AFM) [ 34 ], fluorescence spectroscopy [ 35 ], high-performance liquid chromatography (HPLC) [ 36 ], capillary electrophoresis (CE) [ 4 ], and others.…”

Probing Molecular Packing of Lipid Nanoparticles from 31P Solution and Solid-State NMR

“…28 Doyen et al have measured 31 P solution NMR to evaluate the insertion of peptides into liposomes by observing the change of peak line width. 29 Besides the analysis of phospholipids, Cullis and co-workers have applied 31 P solution NMR to identify the encapsulation of siRNA. 15 In the current study, we determine 31 P chemical shift anisotropy (CSA) of distearoylphosphatidylcholine (DSPC) liposomes across a variety of sample conditions and preparation parameters (i.e., temperature, pH, and size extrusion) to assess the sensitivity and robustness of this parameter in an LNP model system.…”