This study investigates the membrane permeabilization potential of cell-penetrating peptides (CPPs) using large unilamellar vesicles (LUVs). By measuring fluorescence changes, the interaction between CPPs and lipid membranes is analyzed to assess their ability to facilitate cargo delivery.
Cell-penetrating peptides, CPPs, short, positively charged peptides, can interact with and cross cellular membranes, facilitating the delivery of cell-impermeable cargoes.
To study the membrane permeabilization potential of a test CPP in vitro using a cell membrane mimic, begin with a large unilamellar vesicle, LUV, suspension.
LUVs comprise a phospholipid bilayer loaded with a membrane-impermeable fluorescent probe and corresponding quencher pair. Within the vesicles, the quencher interacts with the fluorescent probe due to their close proximity, causing fluorescence quenching.
Pipette the LUV suspension into a quartz fluorescence cuvette, and dilute using buffer. Add a magnetic bar. Transfer to a spectrofluorometer with magnetic stirring to prevent LUV sedimentation during the run. Determine the background fluorescence to eliminate any LUV leaks.
Add the desired concentration of CPP to the LUV suspension at regular intervals.
Based on the hydrophobicity and charge of the CPPs' amino acid residues, they perturb the LUV lipid membrane, leading to membrane penetration and permeabilization.
The vesicle membrane damage causes leakage of the fluorescent probe and quencher into the surrounding solution, increasing their spatial separation. As a result, the probe exhibits fluorescence.
Further CPP solution addition leads to increased LUV permeabilization and fluorescence intensity. An increase in fluorescence intensity with CPP addition suggests the peptide's membrane permeabilizing potential.
To measure the fluorescence leakage, dilute the LUVs in 1 milliliter of HEPES Buffer 2 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% fluorescence leakage as a positive control, add 1 microliter of Triton X-100 to the LUVs to solubilize the vesicles, resulting in a complete unquenching of the probe during the last 100 seconds of the analysis. Then, use the formula to calculate the leakage percentage at each time point.
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