Arp2/3 mediated dynamic lamellipodia of the hPSC colony edges promote liposome-based DNA delivery

Abstract Cationic liposome-mediated delivery of drugs, DNA, or RNA plays a pivotal role in small molecule therapy, gene editing, and immunization. However, our current knowledge regarding the cellular structures that facilitate this process remains limited. Here, we used human pluripotent stem cells (hPSCs), which form compact colonies consisting of dynamically active cells at the periphery and epithelial-like cells at the core. We discovered that cells at the colony edges selectively got transfected by cationic liposomes through Arp2/3 dependent dynamic lamellipodia, which is augmented by myosin II inhibition. Conversely, cells at the core establish tight junctions at their apical surfaces, impeding liposomal access to the basal lamellipodia and thereby inhibiting transfection. In contrast, liposomes incorporating mannosylated lipids are internalized throughout the entire colony via receptor-mediated endocytosis. These findings contribute a novel mechanistic insight into enhancing therapeutic delivery via liposomes, particularly in cell types characterized by dynamic lamellipodia, such as immune cells, or those comprising the epithelial layer. Significance Statement Drug or gene delivery to human cells is essential for effective treatment. Cationic liposomes provide a safe delivery vehicle compared to viruses. However, the cellular structures required for internalizing liposomes are not yet fully understood. Using human stem cells which grow in compact colonies with more dynamic cells at the periphery and epithelial like cells at the center, here we discovered that Arp2/3 dependent dynamic lamellipodia promotes cationic liposome delivery in dynamic cells while receptor mediated endocytosis is required for epithelial cells. This is significant as it provides mechanisms for enhancing liposome delivery to both migratory and epithelial cells in our body. Abstract Figure Graphical Abstract Mechanisms for liposome transfection to the lamellipodial or epithelial cells. Data shown here suggest cationic liposomes fuse with the negatively charged dynamic lamellipodia membrane in an Arp2/3 dependent manner and the process is enhanced by Myosin II inhibition, such as with the stem cell colony edge cells. However, cells more epithelial in nature such as those inside the stem cell colony center do not possess dynamic lamellipodia at the apical surface, rather they form tight junctions which inhibit cationic liposome transfection. Epithelial cells rely on receptor mediated endocytosis in both Myosin II dependent and independent manners to internalize liposomes with lipids that contain ligands for cell surface receptors such as mannose.