Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide

This fluorescence leakage assay is a simple method for addressing membrane destabilization via biomolecules. Using liposomes encapsulated fluorescent dyes, it can be used to compare different membranes interacting peptides. This method is especially useful to investigate membrane destabilization by cell-penetrating peptides in order to understand the mechanism of cellular internalization and to assess their involvement in direct membrane translocation.

Compared to biological membranes, liposomes enable control of the phospholipid composition in order to mimic different lipid values, such as plasma, endosomal, or mitochondrial membranes. To prepare LUVs for their use as cell membrane mimics for a florescence leakage assay, use a Hamilton glass syringe to mix phosphatidylcholine, sphingomyelin, and cholesterol at a 4:4:2 molar ratio. Use a rotary evaporator to evaporate the methanol chloroform under vacuum for 46 to 50 minutes at 60 degrees Celsius until a dried lipid film has formed.

To reconstitute the multi-lamellar vesicles, resuspend the dried lipid film with one milliliter of lipid hydration solution and vortex thoroughly until the dried lipid film has dissolved. After confirming that all of the lipid has been incorporated and that no film is left on the sides of the flask, freeze and thaw the vesicles five times for 30 seconds in liquid nitrogen and two minutes in a 30 degree water bath per cycle. To prepare lipid extruder, insert two HEPES buffer-treated filter supports into each PTFE extruder piece within the metal extruder canister and place a 0.1 micron pore HEPES humidified polycarbonate membrane onto the top of one filter support.

Screw the two metal extruder canisters together and place the assembled extruder into the holder. To check the extruder for leaks or other problems, introduce an empty one milliliter syringe into the appropriate hole at one extremity of the PTFE extruder piece and another one milliliter syringe loaded with one milliliter of HEPES buffer at the other extremity, then deliver the multi-lamellar vesicle sample to the extruder in the same manner and push the sample from one syringe to the other through the polycarbonate membrane at least 21 times to obtain uniform LUVs. To purify the LUVs, add freshly extruded LUVs onto a liquid chromatography column and let the vesicles enter the cross-linked dextran gel.

Continuously add HEPES buffer two to the column and begin eluting approximately two milliliters of HEPES buffer two. The free yellow ANTS and DPX solution will migrate more slowly than the liposomes. After one milliliter has been eluted, start collecting the purified LUVs in 1.5 milliliter tubes.

When the drops of eluent become opalescent, change the tube to recover the LUV-containing fraction. When the drops are no longer opalescent, elute another 500 microliters in a separate fraction before stopping the elution. To determine the LUV size and polydispersity index, indicate the viscosity of the solvent in the polystyrene semi-microcuvette to be used in the experiment and the dynamic light scattering system and add 500 microliters of the LUV solution to the cuvette, then load the cuvette onto the instrument and measure the size distribution in terms of the mean size of the particle distribution and the homogeneity.

To measure the fluorescence leakage, dilute the LUVs in one milliliter of HEPES buffer two in a quartz fluorescence cuvette to a 100 micromolar final concentration and add a magnetic stirrer to facilitate homogenization of the solution during the experiment. Measure the LUVs alone during the first 100 seconds to assess the background fluorescence before measuring the leakage as an increase in fluorescence intensity upon the addition of aliquots of peptide solution over the next 900 seconds. To measure 100%florescence leakage as a positive control, add one microliter of Triton X-100 to the LUVs to solubilize the vesicles, resulting in a complete un-quenching of the probe during the last 100 seconds of the analysis.

Then use the formula to calculate the leakage percentage at each time point. In the absence of peptides, no fluorescence leakage is observed from the LUVs. The addition of WRAP onto the LUVs induces a significant increase in fluorescence, revealing an important LUV leakage and ANTS release.

After 15 minutes, a leakage of approximately 67%is obtained in the presence of 2.5 micromolar WRAP compared to the Triton positive control. In contrast, when WRAP is assembled at the same concentration with small interfering RNA to form peptide-based nanoparticles, the leakage is 1.5 fold weaker compared to the free peptide. When the conjugated Penetratin-iCAL peptide is used, no significant fluorescence increase is detected, whereas the addition of WRAP-iCAL peptide induces a net leakage characterized by very strong fluorescent signal at the same 2.5 micromolar concentration.

These results indicate that the fluorescence leakage assay can be used to reveal the ability of some cell-penetrating peptides to perform peptide membrane interactions leading to a more or less pronounced membrane permeability. This method favors a rapid identification of membrane peptides interactions that lead to membrane destabilization in order to decipher the internalization mechanism of cell-penetrating peptides or other membrane active peptides such as photogenic or antimicrobial peptides. In case of peptide-lipid interaction without leakage induction, other approaches such as tryptophan fluorescence experiments are required.

This fluorescence leakage assay is widely used within the CPP field and can provide important information about cell translocation or endosomal escape.