P–265 Investigating the nanotoxicity of solid silica nanoparticles in gametes following in vitro exposure

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Abstract Study question Do solid silica nanoparticles qualify as a new research tool for the in vitro transfer of compounds into gametes prior to Assisted Reproductive Technology (ART). Summary answer Solid silica nanoparticles (SSNPs) could be used as an intra-gamete delivery system to deliver therapeutic biomolecules into gametes prior to ART. What is known already Sperm-mediated gene transfer (SMGT) results in the production of transgenic embryos; however, the success rate of this technique is low. Nanoparticles are an efficient intra-cellular delivery system in vitro. Naturally cell-secreted nanoparticles are involved in the development of gametes. Mesoporous silica nanoparticles have been shown to carry large amounts of compounds and to interact with gametes without toxic effects, thus providing an alternative to naturally secreted nanoparticles. However, this technique is associated with some limitations, such as the size of these nanoparticles. SSNPs can be synthesised on a smaller nanoscale, thus providing higher potential to penetrate gametes and delivering biomolecules. Study design, size, duration This was an experimental in vitro study that investigated the effects of SSNPs on the motility of boar sperm and the degeneration of hamster oocytes, as determined by ooplasm shrinkage. Participants/materials, setting, methods SSNPs (20 nm) were conjugated with fluorescein diacetate–5-maleimide (FDA5M), a fluorescent protein. FDA5M-labelled SSNPS were incubated with boar sperm (N = 3) at 10 and 30µg/ml/107 sperm for four-hours. Motility parameters were assessed by computer-assisted sperm analysis (CASA). Binding potential was evaluated by fluorescent microscopy. Hamster oocytes (7 oocytes/group) were incubated with FDA5M-labelled SSNPs at 100, 150, and 300µg/ml, for two-hours; ooplasm shrinkage was evaluated. Time/matched control sperm was incubated in phosphate-buffered saline and oocytes in KSOM. Main results and the role of chance Exposure to FDA5M-labelled SSNPs did not affect total or progressive sperm motility (P = 0.6735 and 0.9606, respectively), average-path velocity or straight-line velocity after 4-hours of incubation (P = 0.7459 and 0.8696, respectively) compared to controls. SSNPs at 10 µg/ml significantly increased sperm curvilinear velocity after 1-hour (P = 0.0495) and linearity and straightness after 4-hours (P = 0.0389 and 0.0312, respectively). SSNPs at 30 µg/ml significantly increased sperm linearity after 3- and 4-hours (P = 0.0384 and 0.005, respectively). The proportion of sperm showing green fluorescence was significantly higher in the 30µg/ml dose of SSNPs than the 10µg/ml dose after 4-hours (P &lt; 0.00001). In oocytes, the zona pellucida remained morphologically intact and the ooplasm exhibited green fluorescence. The ooplasm of 42% of the oocytes at 300µg/ml showed ooplasm shrinkage (a sign of degeneration); no oocytes showed shrinkage at doses of 100 and 150µg/ml of SSNPs. The green fluorescence in the sperm head and the ooplasm indicated the ability of SSNPs to spontaneously interact non-invasively with these gametes either by surface association or by cell-internalisation. This could provide a safe and non-invasive intra-gamete delivery system for research purposes and clinical therapy. This system could be used to deliver specific agents into gametes prior to ART to improve outcomes. Limitations, reasons for caution The SSNPs are non-biodegradable; it remains unknown as to how gametes or embryos might react with SSNPs over long time periods. The nanotoxicity of SSNPs has not yet been investigated over the long term. SSNPs have still to be tested with embryos to evaluate their effect on embryonic development. Wider implications of the findings: SSNPs could be functionalised to target the nucleus of mammalian gametes and embryos to act as a carrier for oligonucleotides and genes to correct chromosomal abnormalities and to provide genetic therapy in these gametes and embryos to treat hereditary diseases before intra-uterine transfer. Trial registration number Not applicable