Size-Dependent Effects of Phyllanthus niruri Nanoemulsions on Blood-Testis Barrier Integrity and Cellular Responses in TM4 Sertoli Cells

The therapeutic benefits of Phyllanthus niruri (PN) are well-known, particularly in traditional medicine. Nanoemulsion technology has enhanced its bioavailability and efficacy, but potential nanotoxic effects, especially on male reproductive health, are not fully understood. This study examines the cytotoxic and genotoxic impacts of Phyllanthus niruri nanoemulsions (PNNE) of different sizes on TM4 Sertoli cells, focusing on their effects on the blood-testis barrier (BTB). PNNE of two distinct sizes, were prepared using spontaneous emulsification. The cytotoxicity of these formulations was assessed using a Cell Counting Kit-8 (CCK-8) assay, while genotoxicity was evaluated through an alkaline comet assay. Additionally, the expression of BTB proteins, claudin 11 and connexin 43, was examined via immunofluorescence analysis. PNNE were synthesized with droplet sizes of $16.9~\pm ~3.31$ nm and $163.7~\pm ~8.53$ nm. The smaller PNNE exhibited higher cytotoxicity (IC $50= 160.6~\pm ~8.3~\mu $ g/mL) compared to the larger PNNE (IC $50= 324.4~\pm ~12.5~\mu $ g/mL) and caused more significant DNA damage, as evidenced by the comet assay Both sizes led to a reduction in BTB protein expression, with the smaller nanoparticles causing more pronounced disruption. The study highlights the crucial role of nanoparticle size in determining the biological effects of PNNE on TM4 Sertoli cells. Smaller PNNE were found to be more detrimental to BTB integrity and cellular health, emphasizing the need for careful size optimization in the development of nanoemulsion-based therapies. These findings contribute to the understanding of nanotoxicity in the context of male reproductive health.