Turbidimetric Limulus Amoebocyte Lysate Assay: A Technique to Detect and Quantify Endotoxin Contamination in Nano-Formulated Drugs

Endotoxin, a gram-negative bacterial toxin present on the outer membrane, is a lipopolysaccharide, LPS, composed mainly of lipids and polysaccharides, including lipid A and O-antigen. When released during infection, it is recognized by the immune system, triggering cytokine-based inflammatory responses.

To estimate endotoxin contamination in a liposomal drug nano-formulation using the Limulus amoebocyte lysate, LAL, assay, prepare reaction tubes, one containing the drug and the other containing the drug spiked with a known endotoxin concentration.

Add LAL reagent containing protein lysate obtained from horseshoe crab, Limulus polyphemus, blood cells, or amoebocytes. The lysate includes inactive serine proteases - factor B, factor C, proclotting enzyme - and a gel-clotting protein, coagulogen.

Vortex the tubes and load them into a turbidimeter to measure the turbidity of the solutions.

In the presence of contaminating LPS bound to liposomal drugs, the LPS-sensitive factor C gets autocatalytically activated, which then cleaves and activates factor B that converts the pro-clotting enzyme to clotting enzyme. This activated enzyme cleaves the coagulogen peptide bonds, forming coagulin monomers.

Eventually, the monomers polymerize via interactions between the exposed hydrophobic regions on one's head and another's hydrophobic tail, leading to aggregation and formation of insoluble turbid coagulin gel. The measured turbidity helps determine the endotoxin concentration in the nano-formulation.

Calculated spike recovery from the endotoxin concentrations in the spiked and test samples within the acceptable range indicates no major interference from the nano-formulation, validating the presence of endotoxins in the nano-formulated drug.