RESUMEN
Lipid nanoparticles have been clinically successful in particular recently within the vaccine field, but better tools are needed to analyze heterogeneities at the single particle level to progress drug delivery designs to the next level. Especially, liposomal nanocarriers are becoming increasingly complex e.g. by employing environmental cues for shedding their protective PEG layer, however a detailed mechanistic understanding of how the dePEGylation varies from liposome-to-liposome is still missing. Here we present the development of a fluorescence microscopy based assay capable of detecting the enzyme mediated dePEGylation of individual liposomes. We employ this methodology to understand how enzyme type-, concentration- and incubation time, in addition to liposome size, affects the dePEGylation at the single particle level.
Asunto(s)
Liposomas/química , Nanopartículas , Sistemas de Liberación de Medicamentos , Humanos , Microscopía Fluorescente , PolietilenglicolesRESUMEN
Liposomes are the most used drug delivery vehicle and their therapeutic function is closely linked to their lipid composition. Since most liposome characterization is done using bulk techniques, providing only ensemble averages, the lipid composition of all liposomes within the same formulation are typically assumed to be identical. Here we image individual liposomes using confocal microscopy to quantify that liposomal drug delivery formulations, including multiple component mixtures mimicking Doxil, display more than 10-fold variation in their relative lipid composition. Since liposome function is tightly regulated by the physicochemical properties bestowed by the lipid composition, such significant variations could render only a fraction of liposomes therapeutically active. Additionally, we quantified how this degree of compositional inhomogeneity was modulated by liposome preparation method, the saturation state of the membrane lipid, and whether anti-fouling polyethylene glycol (PEG) conjugated lipids were added to the initial lipid mix or inserted after liposome formation. We believe the insights into the factors governing the degree of inhomogeneity offers the possibility for producing more uniform liposomal drug delivery systems, potentially increasing their therapeutic efficacy.