Determination of nanostructures and drug distribution in lipid nanoparticles by single molecule microscopy

Abstract

Drug loading capacity in nanostructured lipid carriers (NLC) depends on the formation of nanostructures within the lipid matrix. However, investigation of these nanostructures with sizes below the diffraction limit of visible light is quite challenging. Thus, until now the determination of structures and drug distribution within NLCs was not possible. Therefore, we aimed at developing a method to visualize the nanostructures within the lipid carriers. Model NLCs loaded with a lipophilic fluorescent drug mimetic, ATTO-Oxa12, were produced and investigated by single-molecule tracking and localization-based superresolution microscopy. Results revealed spherical ATTO-Oxa12-filled nanostructures with diameters of ∼70nm and 120–130nm, both smaller than the NLC size (∼160nm). The ATTO-Oxa12 diffusion constant was calculated from the single-molecule traces (D⩾1μm2/s) and indicated the distribution of the model drug in the oily component. Together these data suggest the existence of drug-loaded oily nanocompartments, which could fill up to ∼50% of the model NLCs’ volume. In conclusion, a novel tool based on single-molecule microscopy is now available that allows for the precise determination of drug distribution and the characterization of lipid nanostructures, information that is paramount for optimizing lipid nanoparticle formulations.