<em>Ex Vivo</em> Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Citations Over TimeTop 10% of 2013 papers

Abstract

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems. In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8.

Related Papers

• → Impact of technical and assay variation on reporting of hemolysis in stored red blood cell products(2017)16 cited
• → Effects of diethylene glycol butyl ether and butoxyethoxyacetic acid on rat and human erythrocytes(2004)21 cited
• → Limiting the Extent of a Delayed Hemolytic Transfusion Reaction With Automated Red Blood Cell Exchange(2013)20 cited
• → Influence of N,N′-Substituted Piperazines on Cytolysis(2016)3 cited
• → Evaluation of hemolysis during storage of red blood cell concentrates processed by centrifugation and settling method by simple gravity in Burkina Faso(2021)1 cited