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Immunology and Infection

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

Published: March 9th, 2013



1Department of Biomedical Engineering, Vanderbilt University, 2Vanderbilt Institute for Nanoscale Science & Engineering, Vanderbilt University, 3Interdisciplinary Materials Science Program, Vanderbilt University, 4Monroe Carell Jr. Children's Hospital, Vanderbilt University Medical Center, 5Department of Chemical & Biomolecular Engineering, Vanderbilt University, 6Department of Cancer Biology, Vanderbilt University
* These authors contributed equally

A hemolysis assay can be used as a rapid, high-throughput screen of drug delivery systems' cytocompatibility and endosomolytic activity for intracellular cargo delivery. The assay measures the disruption of erythrocyte membranes as a function of environmental pH.

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.

Although there are many potential high-impact therapeutic targets inside the cell, the intracellular delivery of agents poses a significant challenge. Frequently, drugs, especially biologics, are internalized by cells and trafficked into vesicles that either lead to degradation of their contents through the endo-lysosomal pathway, or are shuttled back out of the cell via exocytosis.1 In the latter process, the internal pH of the vesicles is acidified to approximately 5-6, which is the optimal pH for activity of enzymes that function in this compartment, such as lysozyme.2

Recently, a number of materials have ....

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1. Preparation and Sterilization of Buffers and Test Agents

  1. 150 mM NaCl buffer: Dissolve 4.383 g NaCl crystals in 500 ml of nanopure water.
  2. pH Buffers: Prepare phosphate buffers at pH 5.6, 6.2, 6.8, and 7.4 by mixing appropriate amounts of monobasic and dibasic sodium phosphate. If samples are to be tested at lower pH values (i.e. pH < 5.6) then a more appropriate buffer, such as citrate buffer, should be used. Buffer recipes are readily available, and an example reference has been pro.......

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Typically, the agents that exhibit ideal pH-dependent hemolytic behavior have the highest potential for cytosolic delivery of drugs, nucleic acids, or other bioactive molecules. This is exemplified by Agent #1 as portrayed in Figure 2, which exhibits minimal hemolysis at pH 7.4, but a sharp increase in hemolytic behavior at endosomal pH ranges (< 6.5). Some agents may exhibit significant levels of hemolytic behavior at physiological pH ranges (Agent #2 at 40 μg/ml; Figure 2), sugge.......

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pH-responsive polymers or other agents designed for endosomolytic function can be rapidly and effectively screened based on lysis of red blood cells at pH values encountered in the endosome (Figure 1; pH 6.8 - early endosome, pH 6.2 - late endosome, pH 5.6 - lysosome).14-17 pH-dependent hemolysis has been used to screen the ability of carriers to mediate endosomal release of biomacromolecular therapeutics (e.g. peptides, siRNA, ODNs, proteins), and results of this assay can be predict.......

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The authors acknowledge funding through the Department of Defense Congressionally Directed Medical Research Programs (#W81XWH-10-1-0445), National Institutes of Health (NIH R21 HL110056), and American Heart Association (#11SDG4890030).


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Name of the reagent Company Catalogue number Comments (optional)
BD Vacutainer - K2EDTA Vacutainer Tubes Fisher Scientific 22-253-145 For blood collection
BD Vacutainer Blood Collection Needles, 20.5-gauge Fisher Scientific 02-665-31 For blood collection
BD Vacutainer Tube Holder / Needle Adapter Fisher Scientific 22-289-953 For blood collection
BD Brand Isopropyl Alcohol Swabs Fisher Scientific 13-680-63 For blood collection
BD Vacutainer Latex-Free Tourniquet Fisher Scientific 02-657-6 For blood collection
Hydrochloric acid (conc.) Fisher Scientific A144-500 For adjustment of pH of D-PBS.
Triton X-100 Sigma-Aldrich T8787 Positive control
Dulbecco's PBS Invitrogen 14190
Nalgene MF75 Sterile Disposable Bottle-Top Filter Unit with SFCA Membrane Fisher Scientific 09-740-44A
BD 96-well plates, flat-bottomed, tissue culture-treated polystyrene Fisher Scientific 08-772-2C For plate-reading at the end of the assay.
BD 96-well plates, round-bottomed, tissue culture-treated polystyrene Fisher Scientific 08-772-17 For incubation of red blood cells with experimental agents.

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