Spiders of the Loxosceles spp. genus have a wide geographic distribution and are among the main causes of arachnidism in Brazil. Despite advances in venom characterization, fundamental aspects such as its in vivo kinetics and biodistribution remain little understood. This study aimed to investigate the biodistribution of Loxosceles intermedia spider venom through radiolabeling with technetium-99 metastable (99ᵐTc). Radiolabeling efficiency was evaluated using paper chromatography and C18 column, and effectiveness was assessed by SDS-PAGE. Biological activity after radiolabeling was verified through an in vitro sphingomyelinase activity assay and by evaluating dermonecrotic and edematogenic effects in rabbits. The results showed that most venom proteins were efficiently radiolabeled and that the venom remained biologically active after radiolabeling, allowing its distribution to be tracked in mice and rabbits. In both models, the venom concentrated at the injection site and was predominantly biodistributed to the kidneys and bladder. These findings are relevant given the distinct pathophysiological profiles of the animal models used. This is the first study to evaluate, in real time and in live animals, the biodistribution of L. intermedia venom through radiolabeling, and it may provide valuable data for understanding its mechanisms of action and improved the diagnostic of loxoscelism.
