Systemic toxicity and immunotoxicity studies of glucose oxidase-loaded extracellular vesicles derived from lung cancer cells.

INTRODUCTION

Extracellular vesicles (EVs) are naturally occurring nanoparticles secreted by diverse cell types and are increasingly explored as delivery vehicles and immunomodulatory platforms. Rigorous preclinical safety and immunotoxicological evaluation is therefore essential prior to clinical translation. Here, using a modified OECD 423 acute toxicity class method, we assessed the toxicity of EVs loaded with glucose oxidase (GOX) along with T-dependent antibody response (TDAR) analyses in repeated-dose toxicity studies.

METHODS

EVs derived from human adenocarcinomic alveolar basal epithelial cells (A549) were used as enzyme carriers. In the acute toxicity study, three female BALB/c mice received a single (50 mL) intravenous (i.v.) injection of GOX-loaded EVs dispersed in phosphate-buffered saline (PBS) at a concentration of 108 EVs mL-1. In the repeated-dose study, female BALB/c mice (n = 6 per group) were administered GOX-loaded EVs (104-108 EVs mL-1) once daily (50 mL, i.v.) over 28 days. Naïve animals treated with PBS served as vehicle controls. Additional controls included mice receiving keyhole limpet hemocyanin (KLH) as an immunostimulatory reference (a single intraperitoneal dose of 100 mg·kg-1 on day 14) and cyclosporine A as an immunosuppressive control (oral administration of 100 mg·kg-1 on days 1, 7, and 14).

RESULTS

Across all regimens, no mortality or treatment-related clinical deterioration was observed. Animals maintained normal body-weight trajectories, and functional assessments revealed no abnormalities in neuromotor performance or general behavior. Histopathological evaluation of major organs did not identify treatment-associated adverse effects. TDAR analysis demonstrated selective modulation of humoral immune responses after repeated exposure to GOX-loaded EVs. Total circulating IgM levels were significantly elevated on days 21 and 28 (approximately 1.5-fold increase; p = 0.0001) relative to controls, whereas IgG concentrations remained unchanged. GOX-loaded EV treatment induced a chemokine-biased transcriptional response, characterized by upregulation of Cxcl9 and modest increases in Cxcl3, Cxcl16, Ccl5, Ccl12, and Ccl17, while canonical pro-inflammatory cytokines showed minimal changes.

DISCUSSION: GOX-loaded EVs demonstrate a favorable short-term in vivo safety profile and a constrained immunological signature, engaging immune pathways in a context-dependent manner rather than acting as classical immune adjuvants, thereby supporting their potential as controllable immunomodulatory therapeutic platforms.