Delivery and uptake of free and liposome-encapsulated cholesterol-conjugated antisense oligonucleotides in Atlantic salmon sperm: insights from high-resolution imaging

The development of efficient methods for large-scale delivery of antisense molecules to Atlantic salmon (Salmo salar L.) eggs remains a significant challenge, limiting the application of antisense technologies in salmon aquaculture at an industrial scale. This study aimed to establish a proof of concept for a non-invasive, accessible method for delivering antisense oligonucleotides (ASOs) to salmon eggs using sperm cells as a delivery vehicle. Salmon spermatozoa were incubated for 24 h at 4 °C with a fluorescently labeled and cholesterol-conjugated antisense morpholino oligonucleotide (MO) in two formulations: cholesterol-conjugated antisense morpholino free in solution (F-MO) and cholesterol-conjugated antisense morpholino encapsulated into liposomes (L-MO). The binding and internalization patterns of F-MO and L-MO in spermatozoa were evaluated using fluorescence super-resolution microscopy. Both F-MO and L-MO incubations resulted in fluorescent binding in nearly 100% of the sperm cells, with distinct preferences for the head and tail regions of the sperm membrane. Notably, L-MO demonstrated superior internalization into the sperm head, as evidenced by higher fluorescence intensity within the head region compared to F-MO. Fertilization trials confirmed the successful transfer of L-MO to the egg, as indicated by visible fluorescence in the zygote. Moreover, incubation with either MO formulation did not adversely affect fertilization success or embryonic development up to the “eyed stage” under the experimental set-up conditions. In summary, high-resolution microscopy revealed enhanced uptake and internalization of L-MO compared to F-MO, enabling delivery and visualization of fluorescence in the one-cell embryo post-fertilization. These findings highlight the potential of sperm cells as a viable delivery vehicle for non-invasive transfer of MO oligonucleotides to salmon eggs. However, further research is required to optimize this approach and evaluate its scalability. Thus, this study provides a foundation for the development of cost-effective and practical ASO-based technologies for application in salmon aquaculture.