Updated Method for In Vitro Analysis of Nanoparticle Hemolytic Properties
Hemolysis is damage to red blood cells (RBCs), which results in the release of the iron-containing protein hemoglobin into plasma. An in vitro assay was developed and described earlier for the analysis of nanoparticle hemolytic properties. Herein, we present a revised version of the original protocol. In this protocol, analyte nanoparticles and controls are incubated in blood. Undamaged RBCs are removed by centrifugation and hemoglobin, released by the damaged erythrocytes, is converted to cyanmethemoglobin by incubation with Drabkin’s reagent. The amount of cyanmethemoglobin in the supernatant is measured by spectrophotometry. This measured absorbance is compared to a standard curve to determine the concentration of hemoglobin in the supernatant. The measured hemoglobin concentration is then compared to the total hemoglobin concentration to obtain the percentage of nanoparticle-induced hemolysis. The revision includes updated details about nanoparticle sample preparation, selection of nanoparticle concentration for the in vitro study, updated details about assay controls and case studies about nanoparticle interference with the in vitro hemolysis assay.
Key wordsNanoparticles Hemolysis Hemoglobin Red blood cells (RBC)
This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
- 3.ASTM F756-00 (2000) Standard practice for assessment of hemolytic properties of materials. ASTM Int, West Conshohocken, PA. doi: 10.1520/F0756-00
- 4.ASTM E2524-08 (2013) Standard test method for analysis of hemolytic properties of nanoparticles. ASTM Int, West Conshohocken, PA. doi: 10.1520/E2524
- 7.Food and Drug Administration, Center for Drug Evaluation and Research (2005). Guidance for industry: estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers http://www.fda.gov/downloads/drugs/guidances/ucm078932.pdf
- 9.Food and Drug Administration, Center for Drug Evaluation and Research, Center for Veterinary Medicine (2001). Guidance for Industry: Bioanalytical method validation http://www.fda.gov/downloads/Drugs/Guidance/ucm070107.pdf