Characterization of 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine–N‑[Methoxy(polyethylene glycerol)-2000] and Its Complex with Doxorubicin Using Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics

Polyethylene glycol (PEG) lipid nanoparticles (LNPs) spontaneously assemble in water, forming uniformly sized nanoparticles incorporating drugs with prolonged blood clearance compared to drugs alone. Previously, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine–N-[methoxy­(polyethylene glycerol)-2000] (DSPE–PEG₂₀₀₀) and several drug adducts, including doxorubicin, were analyzed by a combination of physical and molecular dynamic (MD) studies. In this study, a complete chemical shift assignment of DSPE–PEG₂₀₀₀ plus or minus doxorubicin was achieved using nuclear magnetic resonance (NMR), one-dimensional selective nuclear Overhauser spectroscopy (1D-selNOESY), NOESY, correlation spectroscopy (COSY), total correlated spectroscopy (TOCSY), heteronuclear single quantum coherence (HSQC), and HSQC–TOCSY. Chemical shift perturbation, titration, relaxation enhancement, and NOESY analysis combined with MD reveal detailed structural information at the atomic level, including the location of doxorubicin in the micelle, its binding constant, the hydrophilic shell organization, and the mobility of the PEG₂₀₀₀ tail, demonstrating that NMR spectroscopy can characterize drug–DSPE–PEG₂₀₀₀ micelles with molecular weights above 180 kDa. The MD study revealed that an initial spherical organization led to a more-disorganized oblate structure in an aqueous environment and agreed with the NMR study in the details of the fine structure, in which methyl group(s) of the stearic acid in the hydrophobic core of the micelle are in contact with the phosphate headgroup of the lipid. Although the molecular size of the LNP drug complex is about 180 kDa, atomic resolution can be achieved by NMR-based methods that reveal distinct features of the drug–lipid interactions. Because many drugs have unfavorable blood clearance that may benefit from incorporation into LNPs, a thorough knowledge of their physical and chemical properties is essential to moving them into a clinical setting. This study provides an advanced basic approach that can be used to study a wide range of drug–LNP interactions.