Generating Giant Membrane Vesicles from Live Cells with Preserved Cellular Properties

Abstract

Biomimetic giant membrane vesicles, with size and lipid compositions comparable to cells, have been recognized as an attractive experimental alternative to living systems. Due to the similarity of their membrane structure to that of body cells, cell-derived giant plasma membrane vesicles have been used as a membrane model for studying lipid/protein behavior of plasma membranes. However, further application of biomimetic giant membrane vesicles has been hampered by the side-effects of chemical vesiculants and the utilization of osmotic buffer. We herein develop a facile strategy to derive giant membrane vesicles (GMVs) from mammalian cells in biofriendly medium with high yields. These GMVs preserve membrane properties and adaptability for surface modification and encapsulation of exogenous molecules, which would facilitate their potential biological applications. Moreover, by loading GMVs with therapeutic drugs, GMVs could be employed for drug transport to tumor cells, which represents another step forward in the biomedical application of giant membrane vesicles. This study highlights biocompatible GMVs with biomimicking membrane surface properties and adaptability as an ideal platform for drug delivery strategies with potential clinical applications.

Figure 1

Flow cytometry analysis and confocal images of GMVs derived from HeLa cells. (a) Two-dimensional (2D) dot plots of side light scattered area (SSC-A) versus forward light scattered area (FSC-A) from flow cytometry exhibit two distinct populations for HeLa cells (blue dots) and GMVs (red dots). The inset image is the bright field image of a single GMV. (b) Confocal images of GMVs stained with FM 4-64 dye. The confocal image (c), 3D image (d), and Z-stack image (e) of a single vesicle all exhibit spherical structure with good symmetry. (f)-(h) Images of GMVs incubated with fluorescein demonstrate good integrity. Scale bar is 20 μm for (b) and 10 μm for (c)-(h).

Figure 2

GMVs exhibit well-preserved membrane properties. Flow cytometry analysis of (a) GMVs and (b) HeLa cells incubated with fluorescein-labeled WGA. Confocal images of CCRF-CEM cells and GMVs demonstrate the similar response to WGA-induced aggregation. Overlay images of bright field and fluorescent images of CCRF-CEM cells (c) before and (d) after incubation with WGA. Mixture of CCRF-CEM cells and GMVs (e) before and (f) after incubation with WGA. The overlay images of GMVs incubated with PE-labeled Mouse anti-Human CD71 antibody (g) or Mouse IgG 2a (h) demonstrate the good recognition capability of CD71 on GMVs. About 10000 events were counted for each sample. Scale bar is 30 μm for (g), (h) and 10 μm for (c)-(f) and the inset figures in (g) and (h).

Figure 3

Modification of GMVs with fluorescein-labeled ssDNA. Flow cytometry analysis and confocal image of GMVs incubated with (a) fluorescein-labeled PolyT10-lipid or (b) fluorescein-labeled ssDNA (L77). (c) GMVs encapsulating fluorescein-labeled L77 were stained with FM 4-64 dye. The Z-stack image (d) and 3D image (e) of a single giant vesicle are also shown. The overlay images of green fluorescence and red fluorescence demonstrate that fluorescein-labeled L77 was mainly located inside GMVs (f). Scale bar is 10 μm.

Figure 4

GMVs as packaging of therapeutic drugs for drug delivery. Flow cytometry of GMVs loaded with curcumin (a). Cell viability of HeLa cells incubated with GMVs loaded with curcumin (0.5 μg mL⁻¹) for 48 h (b). Cell viability of CCRF-CEM cells incubated with GMVs loaded with curcumin (4 μg mL⁻¹) for 24 h (c). About 10000 events were counted for each sample. Scale bar is 10 μm. The statistical significance of the observed differences was analyzed by t tests. Statistical significance was set at p < 0.05 (∗P < 0.05, ∗∗P < 0.01).