n-n--4-xylylenebis(pyridinium) and dipicolinic-acid

We describe fluorescence assays for membrane fusion involving the fusion of liposomes with each other and with cultured cells, fluorescence methods to assess liposome uptake by cells and the intracellular delivery of liposome contents, and assays to evaluate liposome membrane permeability. The Tb/DPA and ANTS/DPX assays monitor the intermixing of aqueous contents of liposomes. The NBD-PE/Rhodamine-PE assay follows the intermixing of liposomal lipids. A variation of this method is suitable for detecting the mixing of the inner monolayers of liposomes. The lipid-mixing assay is also used to study the fusion of cationic liposomes and lipoplexes with cultured cells. The intracellular delivery of liposome contents are monitored, via fluorescence microscopy or flow cytometry, by measuring the release of calcein from the liposome interior, and normalized to cell-associated liposomes quantitated with Rhodamine-PE in the membrane of the same liposomes. The release of liposome contents is monitored by the increase in fluorescence of encapsulated carboxyfluorescein, calcein, or ANTS/DPX, or by the decrease in fluorescence of encapsulated Tb/DPA.

Topics: Animals; Cell Line; Cell Membrane Permeability; Flow Cytometry; Fluoresceins; Humans; Liposomes; Membrane Fusion; Microscopy, Fluorescence; Naphthalenes; Permeability; Picolinic Acids; Pyridinium Compounds; Terbium

Topics: Calcium; Chromatography, Gel; Indicators and Reagents; Kinetics; Liposomes; Membrane Fusion; Models, Biological; Naphthalenes; Picolinic Acids; Pyridinium Compounds; Spectrometry, Fluorescence; Terbium

The kinetics of electrically induced fusion of human erythrocyte ghosts were monitored by the Tb/DPA and ANTS/DPX fluorescence fusion assays. Ghosts were aligned by dielectrophoresis using a 3-MHz 350-V/cm alternating field and were fused by single 15- or 50-microseconds electric field pulses of amplitude 2.5-5.0 kV/cm. Fusion was detected immediately after the pulse. The peak fluorescence change due to fusion was always obtained within 7 s of pulse application, and was highest for a 5.0 kV/cm 15-microseconds pulse. Probe leakage was measured separately and became apparent only 2-3 s after the initiation of fusion. Increasing pulse amplitudes produced higher fusion yields but produced more leakage from the fusion products. 50-microseconds pulses produced less fusion, resulting from a disruption of the dielectrophoretic alignment by fluid turbulence immediately after pulse application. Probe leakage was observed only when pulse application was preceded by dielectrophoresis, suggesting that close membrane positioning allows for additional membrane destabilization caused by the high field pulse. The fluorescence kinetics are interpreted using a simplified model depicting three major types of events: (a) fusion without observable leakage, (b) fusion followed by probe leakage, and (c) contact-related leakage from ghosts which do not undergo contents mixing.

Topics: Cell Fusion; Electric Conductivity; Electrophoresis; Erythrocyte Membrane; Humans; Kinetics; Naphthalenes; Picolinic Acids; Pyridinium Compounds