Endotoxin detection in nanoliposomes using diluted <i>Limulus</i> Amebocyte Lysate and isothermal titration calorimetry

Abstract The increasing use of nanomaterial-based products, such as nanoliposome vaccines, has amplified the need for reliable endotoxin testing, particularly for intravenous applications. Conventional Limulus Amebocyte Lysate (LAL) assays can be compromised by nanomaterials or formulation components that mask endotoxins, highlighting the need for alternative detection strategies. Here, we evaluated isothermal titration calorimetry (ITC) for detecting endotoxins encapsulated in two types of nanoliposomes (NLPs) and assessed the influence of individual NLP components on LAL enzymatic activity and viscosity. NLPs were synthesized in the presence of endotoxins, dialyzed to remove non-bound endotoxins, and characterized using high-performance liquid chromatography (HPLC) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) to confirm endotoxin incorporation. Dynamic light scattering (DLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM) were used to assess size, morphology, and aggregation behavior Endotoxins produce with C 16:0 and C18:1 lipids were found to integrate into NLPs, impeding enzymatic reactions in LAL assays, with loading varying according to NLP type. For ITC detection, NLPs were disrupted using 0.2 % Triton X in a 3:2 nanoliposome-to-detergent ratio followed by brief ultrasonication. Despite the additional time and energy required for ITC experiments, the ITC provides a sensitive alternative to LAL, decreasing reliance on horseshoe crab reagents, visual inspection, and chromophores, while remaining insensitive to turbidity and broadly applicable to both organic and inorganic nanomaterials.