Liposome Model Systems to Study the Endosomal Escape of Cell-Penetrating Peptides: Transport across Phospholipid Membranes Induced by a Proton Gradient

Fatemeh Madani,Astrid Gräslund,Alex Perálvarez-Marín

doi:10.1155/2011/897592

Fatemeh Madani, Astrid Gräslund + Show 1 more

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https://doi.org/10.1155/2011/897592

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Abstract

Detergent-mediated reconstitution of bacteriorhodopsin (BR) into large unilamellar vesicles (LUVs) was investigated, and the effects were carefully characterized for every step of the procedure. LUVs were prepared by the extrusion method, and their size and stability were examined by dynamic light scattering. BR was incorporated into the LUVs using the detergent-mediated reconstitution method and octyl glucoside (OG) as detergent. The result of measuring pH outside the LUVs suggested that in the presence of light, BR pumps protons from the outside to the inside of the LUVs, creating acidic pH inside the vesicles. LUVs with 20% negatively charged headgroups were used to model endosomes with BR incorporated into the membrane. The fluorescein-labeled cell-penetrating peptide penetratin was entrapped inside these BR-containing LUVs. The light-induced proton pumping activity of BR has allowed us to observe the translocation of fluorescein-labeled penetratin across the vesicle membrane.

Highlights

Live cells are protected from the surrounding environment by the cell membrane, which only allows compounds with a small molecular size to pass this barrier into the cell
This new class of peptides was introduced in the late 1980s by the discovery of the human immunodeficiency virus type 1 (HIV-1) encoded Tat peptide [2, 3] and the amphiphilic Drosophila Antennapedia homeodomain-derived 16 amino acid penetratin peptide, which was discovered somewhat later [4,5,6,7]
BR was reconstituted into the large unilamellar vesicles (LUVs) using the detergentmediated reconstitution method

Summary

Introduction

Live cells are protected from the surrounding environment by the cell membrane, which only allows compounds with a small molecular size to pass this barrier into the cell. CPPs are defined as short, water soluble and partly hydrophobic and/or polybasic peptides (at most 30–35 amino acid residues) with a net positive charge at physiological pH [1] This new class of peptides was introduced in the late 1980s by the discovery of the human immunodeficiency virus type 1 (HIV-1) encoded Tat peptide [2, 3] and the amphiphilic Drosophila Antennapedia homeodomain-derived 16 amino acid penetratin peptide (pAntp), which was discovered somewhat later [4,5,6,7]. A significant increase in the fluorescence intensity was observed when the sample was illuminated This result indicates that a pH gradient across the membrane enhances the vesicular escape for the examined fluorescein-labeled CPP (Figure 5(b))

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