Exploring MXene–MSN composite interactions with human hexokinase for safe biomedical use

MXenes and MXene nanocomposites are promising candidates for biomedical applications, yet their interactions with essential enzymes remain poorly understood. Here, we investigated the effects of a pristine MXene (Ti₃C₂Tₓ) and MXene–mesoporous silica nanoparticle (MSN) composites, including amine- (MXene-MSN-NH₂) and carboxyl-functionalised (MXene-MSN-COOH) variants, on human hexokinase (HK), an important glycolytic enzyme. Using circular dichroism and fluorescence spectroscopy, we found that the MXene induced concentration-dependent conformational destabilisation, marked by α-helix loss, β-sheet enrichment, and static quenching indicative of ground-state complex formation. MXene–MSN composites moderated these structural perturbations, while functionalised variants exhibited intermediate effects. Consistent with structural changes, HK activity assays revealed dose-dependent inhibition, with pristine MXene causing the strongest effect (40-60% inhibition at 100–200 µg/mL), followed by MXene-MSN-NH₂ and MXene-MSN-COOH (25-35% inhibition), while MXene-MSN exhibited the weakest impact (≤ 25% inhibition at equivalent doses). Correspondingly, adenosine triphosphate (ATP) measurements in MRC-5 cells demonstrated impaired energy metabolism, with ATP reductions of approximately 31% (MXene), 29% (MXene-MSN-NH₂), 22% (MXene-MSN-COOH), and ≤ 5% (MXene-MSN) at 100 µg/mL, despite maintained cell viability (> 85%). At 200 µg/mL, ATP depletion coincided with moderate reductions in cell viability, indicating progression from sub-lethal metabolic stress to cytotoxicity. These findings establish a clear structure–activity relationship, demonstrating that surface chemistry and silica-based composite formation significantly modulate MXene–enzyme interactions and downstream bioenergetic effects. This work underscores the importance of integrating molecular-level nanosafety assessments to guide the safe design of MXene-based materials for biomedical applications.