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).

1. Preparation and Sterilization of Buffers and Test Agents
<ol>
 <li>150 mM NaCl buffer: Dissolve 4.383 g NaCl crystals in 500 ml of nanopure water.</li>
 <li>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 &lt; 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...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosomal+escape+pathways+for+delivery+of+biologicals.">Endosomal escape pathways for delivery of biologicals.</a> Journal of Controlled Release. 151, 220-228 (2011).</li><li>Plank, C., Oberhauser, B., Mechtler, K., Koch, C., Wagner, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+endosome-disruptive+peptides+on+gene+transfer+using+synthetic+virus-like+gene+transfer+systems.">The influence of endosome-disruptive peptides on gene transfer using synthetic virus-like gene transfer systems.</a> Journal of Biological Chemistry. 269, 12918-12924 (1994).</li><li>Ratner, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+Cells+Are+Sensitive+Detectors+of+Bacterial+Pore-forming+Toxins.">Epithelial Cells Are Sensitive Detectors of Bacterial Pore-forming Toxins.</a> Journal of Biological Chemistry. 281, 12994-12998 (2006).</li><li>Saar, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-penetrating+peptides:+A+comparative+membrane+toxicity+study.">Cell-penetrating peptides: A comparative membrane toxicity study.</a> Analytical Biochemistry. 345, 55-65 (2005).</li><li>Kichler, A., Leborgne, C., Coeytaux, E., Danos, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylenimine-mediated+gene+delivery:+a+mechanistic+study.">Polyethylenimine-mediated gene delivery: a mechanistic study.</a> The Journal of Gene Medicine. 3, 135-144 (2001).</li><li>Behr, J. -. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Applied Phlebotomy. , (2005).</li><li>Bulmus, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+pH-responsive+and+glutathione-reactive,+endosomal+membrane-disruptive+polymeric+carrier+for+intracellular+delivery+of+biomolecular+drugs.">A new pH-responsive and glutathione-reactive, endosomal membrane-disruptive polymeric carrier for intracellular delivery of biomolecular drugs.</a> Journal of controlled release : official journal of the Controlled Release Society. 93, 105-120 (2003).</li><li>Lackey, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemolytic+Activity+of+pH-Responsive+Polymer-Streptavidin+Bioconjugates.">Hemolytic Activity of pH-Responsive Polymer-Streptavidin Bioconjugates.</a> Bioconjugate Chemistry. 10, 401-405 (1999).</li><li>Murthy, N., Campbell, J., Fausto, N., Hoffman, A. S., Stayton, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinspired+pH-responsive+polymers+for+the+intracellular+delivery+of+biomolecular+drugs.">Bioinspired pH-responsive polymers for the intracellular delivery of biomolecular drugs.</a> Bioconjugate chemistry. 14, 412-419 (2003).</li><li>Murthy, N., Robichaud, J. R., Tirrell, D. A., Stayton, P. S., Hoffman, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+design+and+synthesis+of+polymers+for+eukaryotic+membrane+disruption.">The design and synthesis of polymers for eukaryotic membrane disruption.</a> Journal of controlled release : official journal of the Controlled Release Society. 61, 137-143 (1999).</li><li>Yu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+endosomal+barrier+by+amphotericin+B-loaded+dual+pH-responsive+PDMA-b-PDPA+micelleplexes+for+siRNA+delivery.">Overcoming endosomal barrier by amphotericin B-loaded dual pH-responsive PDMA-b-PDPA micelleplexes for siRNA delivery.</a> ACS nano. 5, 9246-9255 (2011).</li><li>Nelson, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+local+delivery+of+siRNA+from+an+injectable+scaffold.">Sustained local delivery of siRNA from an injectable scaffold.</a> Biomaterials. 33, 1154-1161 (2012).</li><li>Miozzari, G. F., Niederberger, P., H&#252;tter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeabilization+of+microorganisms+by+Triton+X-100.">Permeabilization of microorganisms by Triton X-100.</a> Analytical Biochemistry. 90, 220-233 (1978).</li><li>Chen, H., Zhang, H., McCallum, C. M., Szoka, F. C., Guo, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unsaturated+Cationic+Ortho+Esters+for+Endosome+Permeation+in+Gene+Delivery.">Unsaturated Cationic Ortho Esters for Endosome Permeation in Gene Delivery.</a> Journal of Medicinal Chemistry. 50, 4269-4278 (2007).</li><li>Roth, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Measurements+and+Rational+Materials+Design+for+Intracellular+Delivery+of+Oligonucleotides.">Quantitative Measurements and Rational Materials Design for Intracellular Delivery of Oligonucleotides.</a> Biotechnology Progress. 24, 23-28 (2008).</li><li>Blumenthal, R., Seth, P., Willingham, M. C., Pastan, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH-dependent+lysis+of+liposomes+by+adenovirus.">pH-dependent lysis of liposomes by adenovirus.</a> Biochemistry. 25, 2231-2237 (1986).</li><li>Moore, N. M., Sheppard, C. L., Barbour, T. R., Sakiyama-Elbert, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+endosomal+escape+peptides+on+in+vitro+gene+delivery+of+polyethylene+glycol-based+vehicles.">The effect of endosomal escape peptides on in vitro gene delivery of polyethylene glycol-based vehicles.</a> The Journal of Gene Medicine. 10, 1134-1149 (2008).</li><li>Panyam, J., Zhou, W. Z., Prabha, S., Sahoo, S. 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IRB approval: Procedures involving human subjects have been approved by the Vanderbilt University Institutional Review Board (IRB; Protocol #111251). No conflicts of interest declared.

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...

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 been specifically engineered to leverage the acidification of endosomes to facilitate cytosolic delivery of their cargo. One example of this approach uses synthetic, polymer micelle nanoparticles whose core is zwitterionic and charge-neutral at physiologic pH (i.e. 7.4). However, at pH 6.0 - 6.5, the polymers become protonated and acquire a net positive charge that destabilizes the micelle core, and the exposed polymer segments interact with and disrupt the endosomal membrane. This activity has been shown to promote the endosomal escape of peptide and nucleic acid-based therapeutics, allowing them to access their cytosolic targets.3,4 Other examples of methods developed to mediate endosomal escape that disrupt the membrane barrier include 'fusogenic' peptides or proteins that can mediate membrane fusion or transient pore formation in the phospholipid bilayer.5 Homopolymers of anionic alkyl acrylic acids such as poly(propylacrylic acid) are another well-studied approach, and in these polymers, the protonation state of pendant carboxylic acid dictates transition into a hydrophobic, membrane-disruptive state in endo-lysosomal pH ranges.6,7
One useful model system for screening endosomolytic behavior is the ex vivo pH-dependent hemolysis assay.8 In this model system, the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. This generalizable model has been used by others to evaluate the endosomolytic behavior of cell-penetrating peptides and other polymeric gene delivery systems.8-11 In this experiment, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular (7.4), early endosomal (6.8), and late endo-lysosomal (&lt; 6.8) environments. The amount of hemoglobin released during the incubation period is quantified as a measure of red blood cell lysis, which is normalized to the amount of hemoglobin released in positive control samples lysed with a detergent. 
From screening a small library of potentially endosomolytic test materials, one can infer that samples that produce no hemolysis at pH 7.4, but significantly elevated hemolysis at pH &lt; 6.5, will be the most effective and cytocompatible candidates for cytosolic drug delivery. Materials that fit these criteria would be expected to remain inert and not indiscriminately destroy lipid bilayer membranes (i.e. that could cause cytotoxicity) until being exposed to a drop in the local pH following internalization into endo-lysosomal compartments. 
In this protocol, erythrocytes are isolated from a human donor and co-incubated at pH 5.6, 6.2, 6.8, or 7.4 with experimental endosomolytic drug delivery agents. Intact erythrocytes are pelleted, and the supernatants (containing hemoglobin released from lysed erythrocytes) are analyzed for the characteristic absorbance of hemoglobin via a plate reader (Figure 1).

<table border="1">
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>BD Vacutainer - K2EDTA 			Vacutainer Tubes</td>
 <td>Fisher Scientific</td>
 <td>22-253-145</td>
 <td>For blood collection</td>
 </tr>
 <tr>
 <td>BD Vacutainer Blood Collection 			Needles, 20.5-gauge</td>
 <td>Fisher Scientific</td>
 <td>02-665-31</td>
 <td>For blood collection</td>
 </tr>
 <tr>
 <td>BD Vacutainer Tube Holder / Needle 			Adapter</td>
 <td>Fisher Scientific</td>
 <td>22-289-953</td>
 <td>For blood collection</td>
 </tr>
 <tr>
 <td>BD Brand Isopropyl Alcohol Swabs</td>
 <td>Fisher Scientific</td>
 <td>13-680-63</td>
 <td>For blood collection</td>
 </tr>
 <tr>
 <td>BD Vacutainer Latex-Free Tourniquet</td>
 <td>Fisher Scientific</td>
 <td>02-657-6</td>
 <td>For blood collection</td>
 </tr>
 <tr>
 <td>Hydrochloric acid (conc.)</td>
 <td>Fisher Scientific</td>
 <td>A144-500</td>
 <td>For adjustment of pH of D-PBS.</td>
 </tr>
 <tr>
 <td>Triton X-100</td>
 <td>Sigma-Aldrich</td>
 <td>T8787</td>
 <td>Positive control</td>
 </tr>
 <tr>
 <td>Dulbecco's PBS</td>
 <td>Invitrogen</td>
 <td>14190</td>
 <td></td>
 </tr>
 <tr>
 <td>Nalgene MF75 Sterile Disposable 			Bottle-Top Filter Unit with SFCA Membrane</td>
 <td>Fisher Scientific</td>
 <td>09-740-44A</td>
 <td></td>
 </tr>
 <tr>
 <td>BD 96-well plates, flat-bottomed, 			tissue culture-treated polystyrene</td>
 <td>Fisher Scientific</td>
 <td>08-772-2C</td>
 <td>For plate-reading at the end of the 			assay.</td>
 </tr>
 <tr>
 <td>BD 96-well plates, round-bottomed, 			tissue culture-treated polystyrene</td>
 <td>Fisher Scientific</td>
 <td>08-772-17</td>
 <td>For incubation of red blood cells 			with experimental agents.</td>
 </tr>
</table>

ex vivo red blood cell hemolysis assay for the evaluation of ph-responsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs

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.

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 (&lt; 6.5). Some agents may exhibit significant levels of hemolytic behavior at physiological pH ranges (Agent #2 at 40 &mu;g/ml; Figure 2), sugge...

Watch this Scientific Journal Video about Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs at JoVE.com

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

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.

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

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this ...

ex-vivo-red-blood-cell-hemolysis-assay-for-evaluation-ph-responsive

Research

JoVE Journal

Immunology and Infection

33.9K Görüntüleme.  Vanderbilt University. Bir hemoliz tahlil hücre içi kargo için ilaç dağıtım sistemleri 'cytocompatibility ve endosomolitik aktivite hızlı, yüksek hacimli ekran olarak kullanılabilir. Tahlil çevresel pH bir fonksiyonu olarak eritrosit membran parçalama ölçer.

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