Nanotechnology Characterization Lab, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.
Department of Biochemistry, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, P.O.B. 12272, Jerusalem 91120, Israel.
Molecules. 2017 Dec 21;23(1):12. doi: 10.3390/molecules23010012.
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 immunological tests require donor blood. To enable such tests one has to prevent the blood from coagulating, which is usually achieved by the addition of an anticoagulant into blood collection tubes. Heparin, ethylene diamine tetraacetic acid (EDTA), and citrate are the most commonly used anticoagulants. Novel anticoagulants such as hirudin are also available but are not broadly used. Despite the notion that certain anticoagulants may influence assay performance, a systematic comparison between traditional and novel anticoagulants in the in vitro assays intended for immunological characterization of nanotechnology-based formulations is currently not available. We compared hirudin-anticoagulated blood with its traditional counterparts in the standardized immunological assay cascade, and found that the type of anticoagulant did not influence the performance of the hemolysis assay. However, hirudin was more optimal for the complement activation and leukocyte proliferation assays, while traditional anticoagulants citrate and heparin were more appropriate for the coagulation and cytokine secretion assays. The results also suggest that traditional immunological controls such as lipopolysaccharide (LPS ) are not reliable for understanding the role of anticoagulant in the assay performance. We observed differences in the test results between hirudin and 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 anticoagulant and consider individual nanoparticles on a case-by-case basis.
新型纳米技术药物产品的临床前安全性评估通常依赖于体外检测，尤其是在产品开发的早期阶段，因为此类研究可用的纳米材料数量有限。大多数免疫学检测都需要供体血液。为了能够进行这些检测，必须防止血液凝固，这通常通过在血液采集管中添加抗凝剂来实现。肝素、乙二胺四乙酸（EDTA）和柠檬酸盐是最常用的抗凝剂。新型抗凝剂如水蛭素也可用于此类检测，但未被广泛使用。尽管某些抗凝剂可能会影响检测性能，但目前还没有在用于纳米技术制剂免疫学特征描述的体外检测中对传统和新型抗凝剂进行系统比较。我们将含有水蛭素的抗凝血液与传统抗凝剂的血液在标准化免疫检测级联中进行了比较，发现抗凝剂的类型并不影响溶血检测的性能。然而，对于补体激活和白细胞增殖检测，水蛭素更优，而对于凝血和细胞因子分泌检测，传统抗凝剂柠檬酸盐和肝素更合适。结果还表明，传统的免疫对照物如脂多糖（LPS）对于理解抗凝剂在检测性能中的作用不可靠。我们观察到，在没有看到传统对照物产生此类影响时，对于纳米材料，在使用水蛭素和传统抗凝剂制备的血液之间，测试结果存在差异。因此，有必要认识到每种抗凝剂的优缺点，并根据具体情况考虑个体纳米颗粒。
