Characterization of DSPE-PEG2000 and its Complex with Doxorubicin Using NMR Spectroscopy and Molecular Dynamics.

Abstract

Polyethyleneglycol (PEG) lipid nanoparticles (LNPs) spontaneously assemble in water forming uniform sized nanoparticles incorporating drugs with prolonged blood clearance over drugs alone. Previously, 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 NMR 1D-selNOESY, NOESY, COSY, TOCSY, 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 location of doxorubicin in the micelle, its binding constant, the hydrophilic shell organization and the mobility of PEG2000 tail, demonstrating NMR spectroscopy can characterize drug-DSPE-PEG2000 micelles with molecular weights above 180kDa. 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 details of the fine structure where methyl group(s) of the stearic acid in the hydrophobic core of the micelle are in contact with the phosphate head group 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. Since many drugs have unfavorable blood clearance that may benefit from incorporation into LNPs, a thorough knowledge of their physical-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.