Step-wise linking of vesicles by combining reversible and irreversible linkers-towards total control on vesicle aggregate sizes

Small vesicle aggregates as a model for primitive cellular assemblies or for application as multi-compartment drug delivery systems recently received a lot of interest, yet controlling the aggregation of vesicles to predetermined aggregate sizes remains quite a challenge. We show that this type of control is possible by using a combination of two different linker systems: streptavidin-biotin and C18-pNIPAm. The latter linker is a thermoresponsive surfactant, which below its lower critical solution temperature (LCST) of 32 °C acts as barrier on the outside of the vesicles preventing aggregation, even in the presence of other linkers. Above the LCST however, C18-pNIPAm collapses, becomes sticky and thus acts as a linker inducing aggregation. By working at low vesicle concentrations and tuning the C18-pNIPAm/lipid ratio, the aggregation is by design limited. When the temperature drops below the LCST again, the aggregation is reversed. However, this is not the case if other linkers are present. The collapse of C18-pNIPAm above the LCST provides close contact between vesicles, allowing other linker molecules to connect them. By combining the reversible 'switch-like' aggregation properties of C18-pNIPAm, with the irreversible linkage between biotinylated lipids and streptavidin, it is possible to control the size of the aggregates step by step using a simple temperature program.