Figure 1.
Schematic illustration of the preparation and isolation of intact hierarchical unilamellar vesicles (HUVs).
A–C) Preparation of intermediate giant unilamellar vesicles (GUVs) employing the water-in-oil (w/o) emulsion transfer method. Black solid circles indicate biotinylated phospholipids. B, right) Imperfections in either of the two monolayers induce a release of the internal cargo into the hosting solution. C) When passing the water-oil interface the two phospholipid monolayers combine and form a bilayer confining the fluorescently active internal cargo (red circles). D) Repeated extrusion to homogenize the size distribution of the intermediate GUVs. E,F) Preparation of HUVs employing the vesicle-in-water-in-oil emulsion (v/w/o) transfer method. Green solid diamonds indicate fluorescently labeled phospholipids. G, H) Isolation of HUVs from released GUVs using a specially prepared isolation chamber. For details see text.
Figure 2.
Fluorescence micrographs of intact hierarchical unilamellar vesicles (HUVs) and released giant unilamellar vesicles (GUVs).
Intact HUVs and released GUVs before (A–C) and after separation (D–J). A, D, G) Image overlays of the green and red channel micrographs. B, E, H) Separate green channel and C, F, J) separate red channel micrographs. A) The intact HUVs are indicated by the confining membrane fluorescently labeled green and the encapsulated GUVs loaded with a fluorescent cargo (red). In addition, solitary released GUVs not confined by a green labeled membrane are visible. After separation, released GUVs (D) became spatially separated from intact HUVs (G). Scale bar: 25 μm.
Figure 3.
Transmission and fluorescence micrographs of a representative intact hierarchical unilamellar vesicle.
A) The unilamellar fluorescently labeled phospholipid membrane confining the densely packed encapsulated giant unilamellar vesicles (GUVs) is only visible in the fluorescence micrographs B and C. B) Image overlay of C) indicating the envelope membrane labeled green and D) indicating the encapsulated GUVs, the lumen of which is labeled red. Scale bar: 10 μm.
Figure 4.
Size distribution of giant unilamellar vesicles (GUVs) and intact hierarchical unilamellar vesicles (HUVs).
The size distribution of the GUVs is shown in light gray. The size distribution of the HUVs is shown in dark gray. Lines represent normal fits with mean 2.3 μm and standard deviation 0.7 μm (solid line) and mean 9.9 μm and standard deviation 2.6 μm (dashed line).
Figure 5.
Predictions of the minimal and maximal number of encapsulated giant unilamellar vesicles (GUVs).
The minimal number (dashed lines) of encapsulated GUVs needed to induce sedimentation of an intact hierarchical unilamellar vesicle (HUV) of a given size is different for the sedimentation induced by centrifugation (squares) and induced by spontaneous sedimentation (circles). The maximal number (solid line) of encapsulated GUVs that can be packed into an intact HUV of a given size (diamonds) solely depends on the volume available.
Figure 6.
Fluorescence intensity of giant unilamellar vesicles (GUVs) and intact hierarchical unilamellar vesicles (HUVs).
The fluorescence intensity of the lumen of immobilized released GUVs is shown in light gray. The fluorescence intensity of the membrane of intact HUVs is shown in dark gray. Lines represent log-logistic fits with mean 0.42 and standard deviation 0.16 (solid line) and mean 0.13 and standard deviation 0.13 (dashed line).