Cell exposure to hyaluronic acid/ε-Poly-l-Lysine physically crosslinked hydrogel disrupts glutamate metabolism and leads to cytoskeletal collapse

The multifunctional hydrogels made from hyaluronic acid (HA) and ε-poly- l -lysine (PLL) are interesting for soft tissue regeneration and antibacterial use. However, despite their strong antibacterial properties, the biocompatibility of physically crosslinked HA/PLL hydrogel remains inconsistent. This study investigates the molecular mechanisms underlying the cytotoxic effects of HA/PLL hydrogels, particularly their impact on cytoskeletal integrity. Using metabolomic profiling, we identified a marked accumulation of cystine and depletion of glutamate as critical factors. Tracing glutamine metabolism with 13 C-labeled glutamine revealed an inhibition of glutaminase, impairing the conversion of glutamine to glutamate. Gene expression analysis further confirmed the unchanged levels of SLC7A11 gene expression and the downregulation of glutaminase. Additionally, the hydrogels significantly lowered intracellular glutathione levels, disrupting the redox balance. A reduced glutathione level can disturb disulfide bond stability. Confocal fluorescence imaging revealed a disordered and degraded actin. These findings suggest that HA/PLL hydrogels inhibit glutamine metabolism, leading to an elevated cystine/glutamate ratio, resulting in disulfide bond accumulation and subsequent cytoskeletal collapse, characteristic of disulfidptosis. This study provides novel insights into the cytotoxic pathways of HA/PLL hydrogels, with potential implications for their use in antibacterial implants and hydrogel-based therapies in tissue engineering.