Lipopolysaccharide (LPS, endotoxin) is the main component of the cell walls of Gram-negative bacteria and is a very potent initiator of inflammatory reactions in mammalians. It is recognized by most cell types in the body, although immune cells (monocytes, macrophages and dendritic cells) are the most sensitive responders. Endotoxin interaction with specialized cellular receptors, CD14 and Toll-like receptor-4/MD2, results in production of a broad panel of inflammatory cytokines [1
]. The presence of some of these inflammatory cytokines (e.g., TNF-α), may result in tissue damage if produced in large quantities. Others, primarily IL-1β and IL-6, are responsible for febrile reactions and fever [3
]. Owing to the potential for severe immune response, endotoxin levels in pharmaceutical products and medical devices are strictly regulated, with allowable maxima of 5 EU/kg/h for most drug products and 0.2 EU/kg/h for intrathecally administered drugs [4
Drug formulations using nanotechnology are finding increasing application in many areas of medicine, especially for the treatment of cancer. Many nanomedicines for targeted drug delivery of chemotherapeutics have matured beyond the discovery phase of research, and a few such products are US FDA approved and on markets. Such targeted nanodrug delivery systems can minimize dosages, reduce systemic toxicity and reduce adverse side effects of chemotherapeutics, increasing the overall therapeutic efficacy. Endotoxin contamination can be a significant hurdle to the preclinical development of nanoparticle formulations. The large surface areas and high reactivity of nanoparticles make them potential targets for contamination with bacterial endotoxins [5
]. These factors, along with the fact that nanoparticles are frequently synthesized on equipment that may be novel to the pharmaceutical industry, causes endotoxin contamination to be common among many nanoparticle formulations undergoing preclinical characterization [5
]. Such contamination may cause misleading results in toxicity screens (nanoformulations that are not inherently toxic may appear so due to contamination) and in efficacy tests for certain applications (e.g., endotoxin per se
has shown anticancer efficacy). Contamination is often difficult to identify due to nanoparticle interference with traditional assays [6
]. Many conventional (i.e., not nano) pharmaceutical formulations also interfere with tests for endotoxin detection, but the large variation in nanoparticle physicochemical properties causes the spectrum of nanoparticle interferences to be quite wide, making an individual instance of interference difficult to detect.
In recent studies, endotoxin contamination of gold nanoparticles was shown to be associated with undesired inflammatory reactions, while purified gold nanoparticles did not cause an inflammatory response [5
]. Endotoxin itself has been shown to cause tumor regression and was proposed as a drug in clinical oncology trials [7
], although later discontinued due to severe immunotoxicity. These data suggest that endotoxin contamination in nanoparticle formulations intended for cancer therapy may exhibit cytotoxic effects on its own and, therefore, confound results of efficacy studies. Since endotoxin may influence the results of toxicity and efficacy studies, it is important to identify endotoxin contamination before such studies, in order to avoid misinterpretation of study results.
Traditionally, medicine and device pyrogenicity (ability to cause fever, which may or may not be linked to endotoxin levels) is assessed with the in vitro limulus amoebocyte lysate (LAL) assay for endotoxin quantification and the qualitative in vivo rabbit pyrogen test (RPT). The LAL test has three formats: chromogenic, turbidity and gel clot. Here we report that certain types of nanoparticles interfere with one or more of the LAL test formats. Even when the US Pharmacopoeia (USP) formal requirements for validity of the LAL test are met, various formats of the LAL test may produce different results for the same nanoformulation. We show that spike recovery controls (i.e., inhibition enhancement controls prepared by spiking known concentration of endotoxin standard into the nanoparticle formulation under investigation) may help identify interference with a particular LAL test format, although such controls cannot conclusively eliminate the possibility of interference. In situations where the disparity between endotoxin levels obtained by the various LAL formats is large, we found the qualitative in vivo RPT to be useful for verification of LAL findings.