203 Newly designed liposome nanoparticles for drug delivery into boar spermatozoa

Abstract

Liposomes are spherical vesicles with a membrane composed of at least one phospholipid bilayer. They have been successfully applied for intracellular drug or genetic material delivery in many biomedical areas. In agriculture, however, their use to produce transgenic farm animals through exogenous DNA transfection of spermatozoa remains unsatisfactory. Here, we tested a newly designed liposome preparation for effective and harmless interactions with boar spermatozoa. Extended fresh boar semen samples were gently centrifuged (N = 3 independent replicates), and sperm pellets were resuspended in PBS. Sperm concentrations were then adjusted to 2 × 10⁸ mL⁻¹ and aliquoted in 0.5 mL for labelling with various doses of fluorescent liposomes (0, 15, 30, or 60 µg), for plasma membrane labelling (Experiment 1) or with nonfluorescent liposomes loaded with doxorubicin (0 or 60 µg) having natural fluorescence property, for nucleus labelling (Experiment 2). After co-incubation for 45 to 60 min at 37°C, sperm aliquots of each treatment were immediately evaluated for motility and morphology characteristics using a computer-assisted sperm analyzer. Remaining sperm-liposome mixtures were centrifuged to remove the excess of liposomes, and pelleted spermatozoa were imaged with the In vivo Imaging System (IVIS: PerkinElmer, Waltham, MA, USA) and fluorescence confocal laser microscope (LSCM; Zeiss, Oberkochen, Germany). Data were statistically analysed using one-way ANOVA or Student’s t-test, with P < 0.05 indicating a significant difference. In Experiment 1, the proportions of motile and forward progressive spermatozoa were significantly (P < 0.05) increased by the presence of 30 µg (65 ± 4% and 46 ± 3%, respectively) and 60 µg (66 ± 1% and 48 ± 1%, respectively) of liposomes compared with 0 µg, the control (51 ± 3% and 36 ± 3%, respectively). In Experiment 2, doxorubicin-loaded liposomes significantly (P < 0.05) improved the proportions of motile and progressive spermatozoa (87 ± 2% and 69 ± 8% v. 71 ± 8% and 55 ± 8% for the control, respectively). The percentages of static and abnormal spermatozoa (bent tail or distal cytoplasmic droplet) were significantly decreased by the presence of liposomes in both experiments (P < 0.05). In contrast, the velocity parameters of spermatozoa were not significantly affected by the presence of liposomes (P > 0.05). The microscope imaging revealed sperm-liposome interactions with displayed sperm membrane and nucleus fluorescence in Experiments 1 and 2, respectively. The proportion of stained cells in each experiment is in evaluation by flow cytometry. In conclusion, the newly designed liposomes were effective for simple interactions with the plasma membrane and molecule delivery to boar spermatozoa, while improving sperm motility. Further studies are ongoing with the replacement of doxorubicin with a DNA fragment.

This work was supported by USDA-ARS Biophotonics Initiative #58-6402-3-018.