Influence of Environmental Conditions on the Fusion of Cationic Liposomes with Living Mammalian Cells

Abstract

Lipid-based nanoparticles, also called vesicles or liposomes, can be used as carriers for drugs or many types of biological macromolecules, including DNA and proteins. Efficiency and speed of cargo delivery are especially high for carrier vesicles that fuse with the cellular plasma membrane. This occurs for lipid mixture containing equal amounts of the cationic lipid DOTAP and a neutral lipid with an additional few percents of an aromatic substance. The fusion ability of such particles depends on lipid composition with phosphoethanolamine (PE) lipids favoring fusion and phosphatidyl-choline (PC) lipids endocytosis. Here, we examined the effects of temperature, ionic strength, osmolality, and pH on fusion efficiency of cationic liposomes with Chinese hamster ovary (CHO) cells. The phase state of liposomes was analyzed by small angle neutron scattering (SANS). Our results showed that PC containing lipid membranes were organized in the lamellar phase. Here, fusion efficiency depended on buffer conditions and remained vanishingly small at physiological conditions. In contrast, SANS indicated the coexistence of very small (~50 nm) objects with larger, most likely lamellar structures for PE containing lipid particles. The fusion of such particles to cell membranes occurred with very high efficiency at all buffer conditions. We hypothesize that the altered phase state resulted in a highly reduced energetic barrier against fusion.

Keywords: cationic liposomes; fusion conditions; lipid phases; membrane fusion.

Figure 1

3D-fluorescence micrograph of a Chinese hamster ovary (CHO) cell treated with fusogenic liposomes (FLs) DOPE/DOTAP/TFPE-head (1/1/0.1 mol/mol) (A), and endocytic liposomes (ELs) DOPC/DOTAP/TFPE-head (1/1/0.1 mol/mol) (B). Green: TFPE-head signal, red: Nuclei staining with DRAQ5. Scale bar, 20 µm, applies to all.

Figure 2

(A) Fluorescence micrographs of CHO cells treated with fusogenic liposomes (FLs) DOPE/DOTAP/TFPE-head (1/1/0.1 mol/mol), upper row, and endocytic liposomes (ELs) DOPC/DOTAP/TFPE-head (1/1/0.1 mol/mol), lower row. Green: TFPE-head signal, red: Nuclei staining with DRAQ5. Scale bar, 20 µm, applies to all. Experiments were done in PBS buffer. (B) Fusion efficiencies. Whiskers indicate standard deviations of at least three independent experiments. In total, more than 1500 cells were analyzed at each condition.

Figure 3

Scattering curves of DOPE/DOTAP/BODIPY-FL-DHPE (1/1/0.1 mol/mol) (black circles) liposomes and DOPC/DOTAP/BODIPY-FL-DHPE (1/1/0.1 mol/mol) (red circles) liposomes measured at 37 °C. Cyan lines indicate corresponding fits of a single measurement.

Figure 4

(A) Fluorescence micrographs of CHO cells after treatment with fusogenic (DOPE/DOTAP/TFPE-head 1/1/0.1 mol/mol) upper row, and endocytic liposomes, (DOPC/DOTAP/TFPE-head 1/1/0.1 mol/mol) lower row. Green: TFPE-head signal, red: Nucleic staining with DRAQ5. Scale bar, 20 µm, applies to all. (B) Fusion efficiencies. Whiskers indicate standard deviations of at least three independent experiments.

Figure 5

(A) Fluorescence micrographs of CHO cells after treatment with fusogenic (DOPE/DOTAP/TFPE-head, 1/1/0.1 mol/mol), upper row, and endocytic liposomes (DOPC/DOTAP/TFPE-head, 1/1/0.1 mol/mol), lower row, at different pH values. Green: TFPE-head signal, red: Nucleic staining with DRAQ5. Scale bar, 20 µm, applies to all. (B) Fusion efficiencies. Whiskers indicate standard deviations of at least three independent experiments.