Phytogenic bioengineering of chitosan nanoparticles using Flemingia strobilifera (L.) W. T. Aiton: A dual-pronged strategy for Anopheles stephensi control with minimal ecological impact on zebrafish embryos

• Bioengineered F. strobilifera chitosan nanoparticles (ME-CNPs) demonstrated high larvicidal activity against Anopheles stephensi (LC₅₀: 4.60 ppm), a major malaria vector. • A clear No-Observed-Adverse-Effect Level (NOAEL) was established at 75 ppm in zebrafish embryos, indicating a wide safety margin at efficacious larvicidal doses. • At or below the NOAEL, ME-CNPs showed no induction of oxidative stress, apoptosis, or neurotoxicity, confirming their biocompatibility. • The synthesis leverages green principles, resulting in stable, spherical nanoparticles with high encapsulation and controlled release, ideal for sustainable vector control. In this study, chitosan nanoparticles encapsulated with Flemingia strobilifera (L.) W. T. Aiton methanolic extract (ME-CNPs) were synthesized and evaluated for larvicidal activity against Anopheles stephensi and preliminary nanotoxicity using zebrafish embryos. ME-CNPs showed high entrapment efficiency (89.27%) and controlled release (88.75% in 4.5 hours), with a spherical morphology, average size of 54.66 nm, and positive zeta potential (+37.13 mV). The nanoparticles exhibited potent larvicidal activity with an LC₅₀ of 4.60 ppm against 1st instar larvae after 24 hours. In preliminary acute toxicity screening, zebrafish embryos showed no significant apical effects (mortality, malformation, cardiac function) up to 25 ppm. This concentration was operationally defined as the highest tested no-apical-effect level. At higher concentrations (≥75 ppm), dose-dependent toxicity was observed, including increased ROS, apoptosis, reduced AChE activity, and dysregulation of antioxidant enzymes. These findings suggest ME-CNPs possess strong, targeted larvicidal activity and a preliminary margin of safety for apical endpoints in a non-target model at effective concentrations. Further ecotoxicological profiling is warranted to fully assess environmental safety.