PMC

Formation of dextran-loaded GUVs and their exposure to cells. (a) Fluorescence confocal image slices of phase-separated (24 mol% DOTAP, 38 mol% cholesterol, 36 mol% DPPC, 2 mol% PEG2000-DPPE) and homogeneous (24 mol% DOTAP, 74 mol% DOPC, 2 mol% PEG2000-DPPE) GUVs loaded with 20,000 Da TRITC-dextran (red) and labeled with 1 mol% Oregon Green-DPPE (green). (b) Fluorescence confocal image slices of HeLa cells that were incubated with phase-separated 24 mol% DOTAP GUVs (24 mol% DOTAP, 38 mol% cholesterol, 36 mol% DPPC, 2 mol% PEG2000-DPPE) and homogeneous 100 mol% DOPC GUVs loaded with 10,000 Da Rhodamine B-dextran (red) and labeled with Oregon Green-DPPE (1 mol%) (green). All scale bars correspond to 5 µm.

Extraction of cellular blebs confirms the delivery of hydrophilic macromolecules to the cellular cytoplasm. (a) Cartoon illustrating the structure of membrane bound GFP (top), the processes of macromolecular delivery by GUVs (left), and extraction of cellular blebs (right). (b) Fluorescence confocal image slices of GFP expressing HeLa cells (green) that were incubated for 2 h with fusogenic phase-separated GUVs (24 mol% DOTAP, 38 mol% cholesterol, 36 mol% DPPC 2 mol% PEG2000-DPPE) and fusogenic homogeneous GUVs (50 mol% DOTAP, 50 mol% DOPC) loaded with 20,000 Dalton TRITC-dextran (red) and were then induced to release cellular blebs. Arrows indicate dextran-filled blebs at the cellular membrane surface, illustrating that dextran is present in the cellular cytoplasm. All scale bars correspond to 5 µm.

Membrane phase separation promotes lipid mixing in a model membrane assay. (a) Pictorial representation of phase-separated DOTAP containing SUVs fusing to a GUV. (b–e) Fluorescence confocal image slices of 100 mol% DOPC GUVs labeled with 0.3 mol% Oregon Green-DPPE incubated with (b) 100 mol% DOPC SUVs, (c) 100 mol% DOTAP SUVs, (d) Homogeneous 24 mol% SUVs (24 mol% DOTAP, 76 mol% DOPC), and (e) phase-separated 24 mol% SUVs (24 mol% DOTAP, 38 mol% cholesterol, 38 mol% DPPC) labeled with 0.3 mol% Texas Red-DPPE. All scale bars correspond to 5 µm. (f) Bar chart displaying the fraction of the GUV population that exhibited lipid transfer for each SUV composition (n = 3). Error bars correspond to the standard deviation. Brackets show statistically significant comparisons using an unpaired, 2-tailed student’s t test. (g) Bar chart displaying the average fraction of GUVs exhibiting lipid transfer for each PEGylated SUV composition (n = 3). Error bars correspond to the standard deviation. Brackets show statistically significant comparisons using an unpaired, 2-tailed student’s t test.

Phase separation enhances macromolecular delivery to live cells. (a) Flow cytometry histograms showing relative fluorescence of cells incubated with approximately 100 µM (total lipid) of GUVs loaded with 20,000 Da TRITC-dextran. The dashed line is centered on the peak fluorescence (60,000 fluorescence a.u.) of cells exposed to 24 mol% DOTAP 2 mol% PEG2000-DPPE phase-separated GUVs (24 mol% DOTAP, 38 mol% cholesterol, 36 mol% DPPC, 2 mol% PEG2000-DPPE). Each curve represents 3 independent, concatenated trials with a minimum of 5000 cells analyzed per trial. (b) Average red fluorescence in treated and untreated cells (n = 3). Error bars correspond to the standard deviation of all trials. Asterisks demonstrate that all differences between each data were statistically significant (p < 0.02) using an unpaired, 2-tailed student’s t test. The color of the bar corresponds to the legend shown in (a).

Membrane phase separation enhances lipid transfer from liposomes to cells. (a) Flow cytometry histograms showing Oregon Green-DPPE fluorescence for cells incubated with 25, 125, and 250 µM of each SUV condition. The vertical dashed line depicts the peak fluorescence of the untreated cell condition to illustrate the shift in relative Oregon Green-DPPE fluorescence. Each curve represents 3 independent, concatenated trials with a minimum 5000 cells analyzed per trial. (b) Fluorescence confocal image slices of HeLa cells incubated with both homogeneous and phase-separated vesicles. All scale bars correspond to 5 µm. (c, d) The change in average relative fluorescence as a function of increasing concentration of lipid added for cells incubated with (c) phase-separated (5 mol% DOTAP, 10 mol% DOPC, 42.5 mol% cholesterol, 39.5 mol% DPPC, 2 mol% PEG2000-DPPE, 1 mol% Oregon Green-DPPE) and homogenous (5 mol% DOTAP, 92 mol% DOPC, 2 mol% PEG2000-DPPE, 1 mol% Oregon Green-DPPE) 5 mol% DOTAP SUVs, (D) phase-separated (10 mol% DOTAP, 15 mol% DOPC, 42.5 mol% cholesterol, 40.5 mol% DPPC, 2 mol% PEG2000-DPPE, 0.3 mol% Oregon Green-DPPE) and homogenous (10 mol% DOTAP, 88 mol% DOPC, 2 mol% PEG2000-DPPE, 0.3 mol% Oregon Green-DPPE) 10 mol% DOTAP SUVs, (e) phase-separated (24 mol% DOTAP, 38 mol% cholesterol, 36 mol% DPPC, 2 mol% PEG2000-DPPE, 0.3 mol% Oregon Green-DPPE) and homogenous (24 mol% DOTAP, 74 mol% DOPC, 2 mol% PEG2000-DPPE, 0.3 mol% Oregon Green-DPPE) 24 mol% DOTAP SUVs. Each data point represents the average of 3 independent trials per condition. Error bars correspond to the standard deviation. An unpaired, 2-tailed student’s t test was performed to determine statistical significance between homogeneous and phase-separated groups. Asterisks denote statistical significance p < 0.01.

DOTAP can be integrated into a phase-separated membrane system. (a) Molecular structure of lipids. DOTAP (Top), DPPC (Bottom), cholesterol (Right). (b) A ternary DOTAP/DPPC/cholesterol phase diagram that plots the phases present at room temperature (25 °C) for multiple mixtures of DOTAP, DPPC, and cholesterol. All data superimposed over the published phase diagram for the DOPC/DPPC/cholesterol system. The phase boundaries of this diagram are marked with dashed lines because the phase regions of the DOTAP/DPPC/cholesterol system are likely not identical to the DOPC/DPPC/cholesterol system owing to the physio-chemical differences between the headgroups of DOTAP and DOPC. Closed circles indicate points where confocal fluorescence microscopy images were taken. A trace amount (0.3 mol%) of Texas Red-DPPE was used to visualize contrast between phases. As discussed in the text, composition 1 displays a uniform solid phase. Compositions 2 and 3 display a single uniform liquid phase. Composition 4 displays liquid–solid phase coexistence. Composition 5 displays liquid–liquid phase coexistence where the liquid ordered phase makes up the majority of the vesicle. Composition 6 displays liquid–liquid phase coexistence where the liquid disordered phase makes up the majority. All scale bars correspond to 5 µm. (c) Bar chart displaying the average fraction of the highly fluid phase as a function of the molar concentration of DOTAP in the mixture. Error bars represent standard deviation. The area fraction of the domain of at least 16 vesicles were measured per data point. Brackets show statistically significant comparisons using an unpaired, 2-tailed student’s t-test. All p values < 0.003. (d) Confocal fluorescence microscopy slices of phase-separated 24, 16, and 8 mol% DOTAP GUVs, each with a 1:1 ratio of cholesterol to DPPC, indicating decreased area fraction of the disordered phase. All scale bars correspond to 5 µm.