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

The overall goal of this experiment is to quantitatively assess the membrane disrupting potential of drug delivery vectors for the intracellular delivery of biologics with red blood cells serving as a biological model of the endosomal membrane. This video was produced by the Advanced Therapeutics Laboratory at Vanderbilt University. All procedures involving human subjects have been approved by Vanderbilt's Institutional Review Board.

First, a brief introduction to endo, hemolysis and hemolysis. Carriers of biologics are taken into endosomes by the cell through non-specific or receptor mediated endocytosis. Following endocytosis.

The cargo is either degraded in lysosomes or trafficked for exocytosis back into the extracellular space. As shown, however, the decreasing pH in an endosome can be utilized with pH responsive drug carriers that since the acidic environment and become membrane disrupting, physically por rating and or destabilizing the endosomal membrane with the endosome disrupted the drug cargo may be released into the cytoplasm and diffused to the target locations in the cytoplasm, mitochondria, or nucleus. In this assay, red blood cells are used as a model of the endosomal membrane at physiologic pH.

Ideal delivery vehicles will remain stable and will not disrupt the red blood cell membrane. However, upon decreasing pH, the delivery vehicle will become unstable, resulting in hydrophobic interactions that destroy the red blood cell membrane releasing hemoglobin. The hemoglobin may then be isolated from the red blood cells and spectra photometrically quantified by absorbance at 405, 450 or 541 nanometers.

Higher absorbence values will correlate to more potent endom lytic behavior. This can be visualized by time-lapse microscopy at physiologic pH. The red blood cells remain stable in the presence of the drug carrier.

However, at a lower pH characteristic of the endosomal pathway, the delivery vehicles rapidly disrupt and destroy the red blood cell membranes. The materials needed for this study include 150 millimolar sodium chloride, prepared with 500 milliliters of deionized water and 4.383 gram sodium chloride sterile filtered through a bottle top vacuum filtration apparatus. Second phosphate buffered saline solutions at pH 7.4, 6.8 6.2, and 5.6 were prepared by mixing the proper amounts of mono and diba sodium phosphate buffers and sterile filtered using a bottle top vacuum filtration apparatus.

The pH was monitored with a pH meter and or pH strips, used several methods to ensure accuracy of measurements. 20%Triton X was made by dissolving 20 milliliters of Triton X 102 80 milliliters of nano pure water shake vigorously and sonicate to dissolve after institutional review board approval. Have your study nurse or phlebotomist collect 25 milliliters of whole blood from an anonymous donor into potassium EDTA coated vacu containers to prevent coagulation.

In this video, a 20.5 gauge collection needle connected to a vacutainer adapter is used. The collected blood is centrifuged at 500 times gravity for five minutes. After the centrifugation, the plasma layer and red blood cell layer should be evident.

Be careful not to disturb the layers by shaking. Mark the plasma levels then aspirate gently with a micro pipetter or vacuum aspirator and discard into bleach. Replace the plasma with 150 millimolar sodium chloride filling to the marked line.

Cap the vial and invert several times to gently mix centrifuge for the second time at 500 G for five minutes, followed by supernatant aspiration. Replace the supernatant to the marked line with 150 millimolar sodium chloride and invert several times to gently mix centrifuge for a third time at 500 G for five minutes. Aspirate the S natin and replace to the marked line with PBS of pH 7.4.

Split the bloodstock evenly into four tubes. One for each pH to be tested, including physiologic pH of 7.4 early endosomal pH of 6.8, late endosomal pH of 6.2 and later endosome or lysosomal pH of 5.8. Repeat centrifugation for fourth and final time at 500 G for five minutes.

Mark the super natan levels and aspirate gently discarding the super natan into bleach. Replace the super natan with PBS of appropriate pH. The final stocks of red blood cells are made by diluting stock solutions 50 times into buffer of the appropriate pH.

In this case, one milliliter of concentrated red blood cells was added to 49 milliliters of PBS gently invert the vial several times to mix red blood cells before blood preparation. Prepare stock solutions of delivery vehicles at 20 times concentration. For polymers, we suggest 2100 and 800 micrograms per milliliter as initial testing concentrations once diluted, the final concentration will be one five and 40 micrograms per milliliter for incubation, use conical or round bottom plates so that intact red blood cells can be removed through centrifugation pipette 10 microliters of 20 times polymer solution into each well.

We suggesting samples in at least triplicate or quad duplicate to optimize results. Triton X will lice all cells and can be used as a positive control at a final concentration of 1%PBS at pH 7.4 can be used as a negative control pipette 190 microliters of diluted red blood cells. To each well ensure red blood cell solution is kept homogenous, which may require slight shaking or mixing to keep them suspended.

Note that once red blood cells are added, you may already see lysis beginning to occur for extremely hemolytic compounds. Incubate for one hour at 37 degrees Celsius to allow lysis to occur after one hour. Remove the plates for centrifugation centrifuge the plates at 500 G to pellet red blood cells.

Then carefully transfer 100 microliters of the supernatant into a flat bottom 96 well plate. It is critical not to disturb the red blood cell pellet, so make sure to handle with care. Measure the absorbance of each well with a plate reader at 450 nanometer.

If hemoglobin is too concentrated and high ABSORBENCE readings are obtained. Plates can also be read at 541 nanometer. Alternatively, if hemoglobin is too dilute and low, absorbence readings are obtained, plates can be read at 405 nanometers to maximize absorbence To analyze the data percent homolysis can be calculated by assuming that PBS will not lyce any red blood cells and that Triton X will ice 100%of the red blood cells.

The equation shown will yield percent homolysis for each sample. Software like Microsoft Excel can used to rapidly analyze experiments from hundreds of wells. Here are representative results at several concentrations over the pH ranges discussed.

This formulation shows an ideal pH response and that it demonstrates no hemolytic activity at physiologic pH, but shows switch like behavior at endosomal pH that results in significant membrane disruption as shown by the red arrow. You've just watched our lab's video describing the pH dependent red blood cell hemolysis assay. This is a relatively inexpensive, easy to use and high throughput assay that can be used to screen combinatorial libraries of series of polymers that are designed for inso lytic delivery of biologic drugs.

We found this assay to be highly predictive of cytosolic delivery and bioactivity of a range of biologics, including proteins, peptides, and nucleic acids. However, it should be mentioned that there are some potential weaknesses of this assay. It's thought to be a good predictive tool for polymers or peptides that actually physically disrupt the endosomal membrane.

However, it is not a good predictive tool to be used for polymers or other systems that act by a proton sponge based mechanism. In addition, one must consider that the makeup of the re of the lipid bilayer of red blood cells does not exactly mimic that of the endosomal or lysosomal membranes. In terms of the lipid composition and cholesterol content, one should always be prepared to follow up.

Any red blood cell hemolysis result with complimentary techniques and assays. For example, fluorescent microscopy used in conjunction with endosomal or lysosomal dyes can be used in colocalization studies to visualize the scape of biologic drugs from the endo lysosomal pathways. In addition, measurement of the bioactivity or downstream effects of the drug or biologic delivered should always be the final step.

Finally, remember to always handle and dispose of biohazardous waste properly as defined by your home institution. That concludes our video. Best of luck in your experiments.