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MONITORING SPERM-LIPOSOME INTERACTIONS WITH RESONANCE ENERGY TRANSFER, FLUORESCENCE DEQUENCHING, AND FLOW CYTOMETRY.
Anzar, Muhammad1, Somnapan-Kakuda, Nipa1, He, LiWei1, Pauls, Karl2, Buhr, Mary1, 1 2
ABSTRACT- Liposomes can be used to carry hormones, anti-cancer agents, enzymes and exogenous DNA into cells, or to alter the lipid composition of cell membranes. The present study was conducted to monitor the fusion between liposomes and mammalian spermatozoa using resonance energy transfer, fluorescence dequenching and flow cytometry (FC) techniques. Experiment 1: Bull sperm were incubated in TALP (35°C, 5 mM Ca++) with PC/PE liposomes containing NBD-PE/Rh-PE (49.7:49.7:0.15:0.45 mol:mol). NBD fluorescence increased, indicating that liposomes were fusing to sperm, allowing the two dyes to spread apart and preventing transfer of energy from NBD to Rh. Fusion was higher with 40 vs 20 x 106 sperm/ml. Experiment 2: Boar sperm (10 and 100 million/ml) were incubated with boar sperm membrane lipids (pML) or defined lipids (DL) of PC/PE/Sph/PS/PI (22:26:42:5:5) at 35°C for 90 min. Liposomes (0.3119 mol/ml) contained 2 mol% of the self-quenching R18 fluorophore. R18 fluorescence increased significantly over time (1min, 25%; 90min >60%; P<0.05), as liposomes fused to boar spermatozoa and the R18 dispersed through the sperm membranes, preventing its self-quenching. Fusion was higher with 100 million sperm/ml, and with pML than DL until 90 min. Experiment 3: Sperm (n=3 bulls, 5 million/ml in TALP, 35°C) were incubated with liposomes (500 g/ml) prepared from L-carnitine, sphingomyelin and phosphatidylinositol (1:2:2) + 2 mol% R18, at 35°C. FC analysis indicated >85% spermatozoa fused with liposomes, varying among bulls (84.9±1.8 = 85.3±1.2 < 88.8±1.1, P<0.05). After 60 min incubation, approximately 75% of available liposomes were fused with sperm. In conclusion, liposomes can fuse with mammalian sperm, with fusion efficiency dependent upon sperm:liposome ratio and liposome composition. Various fluorescence techniques can detect fusion, with flow cytometry best able to quantitate sperm incorporating lipids and the percentage of liposomes that are fused.
KEY WORDS: membranes, modification, lipids, fusion
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