Microfluidic Preparation of Liposomes to Determine Particle Size Influence on Cellular Uptake Mechanisms

Purpose This study investigates the cellular uptake and trafficking of liposomes in Caco-2 cells, using vesicles with distinct average diameters ranging from 40.6 nm to 276.6 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 for liposomes with mean diameters ranging from 40.6 nm to 276.6 nm. When testing these liposomes against endocytosis inhibitors, cellular uptake of the largest (97.8 nm and 162.1 nm in diameter) liposomes were predominantly subjected to clathrin-dependent uptake mechanisms, the medium-sized (72.3 nm in diameter) liposomes seemed to be influenced by all investigated pathways and the smallest liposomes (40.6 nm in diameter) primarily followed a dynamin-dependent pathway. In addition, the 40.6 nm, 72.3 nm, and 162.1 nm diameter liposomes showed slightly decreased accumulation within endosomes after 1 h compared to liposomes which were 97.8 nm in diameter. Conversely, liposome co-localization with lysosomes was consistent for liposomes ranging from 40.6 nm to 97.8 nm in diameter. Conclusions 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.