Engineered acetylcholinesterase-loaded dissolvable microneedles mitigate dermal toxicity by targeting trichlorfon...

Trichlorfon (TCF), a widely used organophosphorus pesticide for crop protection, poses severe dermal toxicity risks as it readily penetrates the epidermal barrier and metabolizes into the more toxic dichlorvos (DDVP), triggering neurotoxicity, oxidative stress and inflammatory damage in exposed individuals. As a promising stoichiometric bioscavenger for pesticide detoxification, natural acetylcholinesterase (AChE) is still plagued by bottlenecks in thermal stability and transdermal delivery efficiency. To address these issues, this study employed a previously engineered high-activity and thermostable AChE variant (CpA-M5), evaluated its detoxification efficacy and mechanisms both in vitro and in vivo, and further developed a CpA-M5-loaded dissolvable microneedle system (CpA-M5-MN) for targeted dermal detoxification. In vitro assays demonstrated that CpA-M5 effectively restored endogenous AChE activity, scavenged ROS, reduced inflammation and alleviated apoptosis in TCF-exposed cells. Enzyme kinetics and molecular interaction analyses confirmed irreversible binding between CpA-M5 and TCF/DDVP, with DDVP showing higher binding affinity (K _D  = 9.23 × 10^-6 M, Ki = 15.56 μM) and complex stability (K _a  = 2450.191 M^-1, ΔG = -39.37 ± 1.48 kcal/mol). In vivo, the CpA-M5-loaded microneedle system achieved targeted transdermal delivery, effectively overcoming the limitations of low permeability and rapid degradation of free enzymes. Pathological and metabolomic analyses demonstrated that CpA-M5-MN reduced skin residual TCF/DDVP levels, restored local oxidative-inflammatory homeostasis, and regulated glutathione and amino acid metabolic pathways, thereby achieving synergistic detoxification and metabolic repair effects. Collectively, this work presents a promising targeted detoxification platform for pesticide-induced dermal injury, and provides new insights into the clinical translation of engineered enzyme-based bioscavengers.