Microfluidic preparation of liposomes to determine particle size influence on cellular uptake mechanisms
Wyatt N. Vreeland, Abhay U. Andar, Renee R. Hood, Don L. DeVoe, Peter W. Swaan
Purpose: This study investigates the cellular uptake and trafficking of liposomes in Caco-2 cells, using vesicles with distinct average diameters ranging from 40 nm-276 nm. Liposomes were prepared by microfluidic hydrodynamic flow focusing, producing nearly-monodisperse populations and enabling size-dependent uptake to be effectively evaluated. Methods: Populations of PEG-conjugated liposomes of various distinct sizes were prepared in a disposable microfluidic device using a simple continuous-flow microfluidic technique. Liposome cellular uptake was investigated using flow cytometry and confocal microscopy. Results: Liposome uptake by Caco-2 cells was observed to be strongly size-dependent. The largest liposomes (97 nm 276 nm) were predominantly subjected to clathrin-dependent uptake mechanisms. Nearly all investigated pathways influenced liposomes within the size range 64 nm 72 nm, and the smallest liposomes (40 nm 50 nm) followed primarily dynamin-dependent pathways. The 40 nm, 72 nm and 162 nm liposomes showed slightly decreased accumulation in endosomes after 1 h compared to the 97 nm liposomes. Conversely, liposome co-localization with lysosomes was consistent for liposomes ranging across sizes 40 nm 97 nm. Conclusion: The continuous-flow synthesis of nearly-monodisperse populations of liposomes of distinct size via a microfluidic hydrodynamic flow focusing technique enabled unique in vitro studies in which specific effects of particle size on cellular uptake were elucidated. The results of this study highlight the significant influence of liposome size on cellular uptake mechanisms and may be further exploited for increasing specificity, improving efficacy, and reducing toxicity of liposomal drug delivery systems.