Fluorescent Lipids: Functional Parts of Fusogenic Liposomes and Tools for Cell Membrane Labeling and Visualization

Abstract

In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traffic processes. We tested the fluorescent derivatives of the following essential membrane lipids for membrane fusion: Ceramide, sphingomyelin, phosphocholine, phosphatidylinositol-bisphosphate, ganglioside, cholesterol, and cholesteryl ester. Our results show that all probed lipids could more efficiently be incorporated into the plasma membrane of living cells than by using other methods. Moreover, labeling occurred in a gentle manner under classical cell culture conditions reducing cellular stress responses. Staining procedures were monitored by fluorescence microscopy and it was observed that sphingolipids and cholesterol containing free hydroxyl groups exhibit a decreased distribution velocity as well as a longer persistence in the plasma membrane compared to lipids without hydroxyl groups like phospholipids or other artificial lipid analogs. After membrane staining, the fluorescent molecules were sorted into membranes of cell organelles according to their chemical properties and biological functions without any influence of the delivery system.

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

“…To prepare the liposomes required for lipid incorporation 36 , dissolve separately 1 mg of DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), 1 mg of DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), and 1 mg of PPE-ATTO488 in 1 ml of chloroform. Mix together 0.5 ml of DOPE solution, 0.5 ml of DOTAP solution, and 25 μl of PPE-ATTO488 solution and dry under vacuum for 24 h. Add 0.5 ml of HEPES buffer 20 mM, vortex for 15 min and sonicate for 15 min at 40 °C.…”

Liposomes in tissue engineering and regenerative medicine

“…Liposomes carrying encapsulated fluorophores have been synthesized as novel fluorescent markers to image flow profiles in micro-fabricated structures [19]. Fluorescent-labelled lipids are incorporated within the lipid bilayer, and they have the following function: trigger rapid membrane fusion, enable fluorescence imaging of cell membranes and membrane traffic processes [50]. Moreover, negatively and positively charged lipids are also added to the liposome formulation to help in the liposome stabilization and avoid agglomeration [51].…”

Tat peptide and hexadecylphosphocholine introduction into pegylated liposomal doxorubicin: An in vitro and in vivo study on drug cellular delivery, release, biodistribution and antitumor activity

“…2B). Since DiI is a lipophilic dye restricted to only membrane of liposomes and cell, the only way for delivery of DiI to cell obtains by direct delivery of liposome to cell (Kleusch et al, 2012). We also measured the Dox fluorescence of the media treated with Dox-liposomes and found the same trend as Dox in cells (data not shown).…”