Reducing endotoxin contamination in chitosan: An optimized purification method for biomedical applications

Chitosan, a natural biopolymer, is widely used in biomedical applications due to its biocompatibility. It also possesses immunostimulatory properties and is notably studied and used as a vaccine adjuvant. However, endotoxin contamination, an often-overlooked extrinsic factor, may compromise its safety and confound the interpretation of its biological or immunostimulatory effects. Despite its widespread use, data on endotoxin levels in commercially available chitosan remain limited, potentially contributing to inconsistent immunological outcomes. In this study, we developed a simple four-step purification process that effectively reduces endotoxin levels without altering key chitosan properties, namely the degree of deacetylation (DDA) and molar mass (MW). We characterized the endotoxin content of various commercial chitosan samples using the Limulus Amebocyte Lysate (LAL) assay and evaluated their immunostimulatory properties in vitro, both before and after purification. A 1 N sodium hydroxide treatment for 48 h reduced endotoxin levels by up to 100-fold, lowering the most contaminated sample from 62,000 EU/g to approximately 600 EU/g, while preserving DDA and MW. In vitro immunoassays showed that proinflammatory cytokine responses (IL-1β, TNF-α, IL-6) in J774A.1 macrophages were primarily driven by endotoxin contamination, which was significantly reduced following purification. Structural properties of chitosan showed no measurable impact on cytokine profiles. This study underscores the critical need for endotoxin assessment and removal in chitosan intended for biomedical applications.