Abstract
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.
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Acknowledgment
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.
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Neun, B.W., Ilinskaya, A.N., Dobrovolskaia, M.A. (2018). Updated Method for In Vitro Analysis of Nanoparticle Hemolytic Properties. In: McNeil, S. (eds) Characterization of Nanoparticles Intended for Drug Delivery. Methods in Molecular Biology, vol 1682. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7352-1_9
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DOI: https://doi.org/10.1007/978-1-4939-7352-1_9
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