Previous studies have reported the pearling instability of GUV nanotubes when the membrane is subject to tension caused by bilayer asymmetry. In our experiments, it is unclear whether LCNP fusion into the GUVs creates bilayer asymmetry or not. Asymmetry exists in terms of the LCNP interaction with the GUV membrane, which is exclusively from the extra-vesicular side of the membrane. However, the rate of MO flip-flop across the bilayer is unknown and any potential asymmetry cannot be resolved from our fluorescence microscopy methods. Lipid flip-flop may occur through collective formation of transmembrane pores, which we often observe after a lag time following GUV growth. Pearling instabilities were mostly observed in unleaked GUVs. Therefore, we speculate that asymmetric membrane stresses from enhanced MO localisation in the outer membrane monolayer of GUVs drives the pearling of lipid tubules that are observed.