In vitro cytotoxic and genotoxic effects of Phyllanthus niruri extract loaded chitosan nanoparticles in TM4 cells and their influence on spermatogenesis

Abstract

Purpose: This paper introduces a complete in vitro investigation of cytotoxic and genotoxic effects of Phyllanthus niruri extract loaded chitosan nanoparticles (PNNP) on mouse Sertoli cell line (TM4), as well as their impact on spermatogenesis. Methods: Chitosan nanoparticles (ChNP) and PNNP were prepared using an ionic gelation process, while their cytotoxicity on TM4 cells was assessed using the Cell Counting Kit-8 (CCK-8) assay. Comet and fast halo assays were used to quantify single-strand DNA breaks in TM4 cells. To detect changes in cell morphology during apoptosis, nuclear staining with Hoechst 33342 was performed. An immunofluorescence assay was employed to examine the expression level of proteins connexin 43 and Claudin 11 in TM4 cells after exposure to PNNP concentration of 125 µg/mL. Results: The synthesized PNNP had a size of 170.6 nm, a polydispersity index of 0.269, a zeta potential of +37.8 mV, and a good entrapment efficiency of 71.0%. Encapsulation of Phyllanthus niruri into ChNP induced DNA damage in TM4 cells as determined by alkaline comet and fast halo assay (FHA). Additionally, it stimulated apoptosis, as determined by changes in cell morphology by Hoechst 33342 staining. There was significant down regulation of blood-testis barrier (BTB) proteins in TM4 cells after exposure to PNNP which could compromise the integrity of BTB and subsequently disrupt spermatogenesis process in male. Conclusion: Our investigation confirms the cytotoxic and genotoxic effects of PNNP in TM4 cells, which could lead to spermatogenesis disruption and male infertility.