Anticoagulants Influence the Performance of In Vitro Assays Intended for Characterization of Nanotechnology-Based Formulations

The preclinical safety assessment of novel nanotechnology-based drug products frequently relies on in vitro assays, especially during the early stages of product development, due to the limited quantities of nanomaterials available for such studies. The majority of immunologicaltests require donor blood. To enable such tests one has to prevent the blood from coagulating, which is usually achieved by the addition of an anticoagulantinto blood collection tubes. Heparin, ethylene diamine tetraacetic acid (EDTA), and citrate are the most commonly used anticoagulants. Novel anticoagulantssuch as hirudinare also available but are not broadly used. Despite the notion that certain anticoagulantsmay influence assayperformance, a systematic comparison between traditional and novel anticoagulantsin the in vitro assaysintended for immunologicalcharacterization of nanotechnology-based formulations is currently not available. We compared hirudin- anticoagulatedblood with its traditional counterparts in the standardized immunological assaycascade, and found that the type of anticoagulantdid not influence the performance of the hemolysis assay. However, hirudinwas more optimal for the complement activation and leukocyte proliferation assays, while traditional anticoagulantscitrate and heparin were more appropriate for the coagulation and cytokine secretion assays. The results also suggest that traditional immunologicalcontrols such as lipopolysaccharide (LPS ) are not reliable for understanding the role of anticoagulantin the assayperformance. We observed differences in the test results between hirudinand traditional anticoagulant-prepared blood for nanomaterials at the time when no such effects were seen with traditional controls. It is, therefore, important to recognize the advantages and limitations of each anticoagulantand consider individual nanoparticles on a case-by-case basis.