Mobility and Translocation of TAT Peptides in Model Membranes

Abstract

Cell penetrating peptides (CPPs) like HIV1-TAT have the special property to traverse the cell membrane and to function as vectors for various macromolecular cargoes such as fluorophores, nucleotides, drugs, proteins, DNA, and peptide-nucleic acids, and even liposomes and magnetic nanoparticles. In spite of the fact that TAT peptides were intensively investigated, the exact internalization mechanism is still controversial. Despite the controversy and uncertainty regarding the uptake mechanism, the property of TAT to deliver non-permeable molecules into living cells makes it an attractive tool for biological sciences as well as medicine and biotechnology. It is therefore essential to identify precisely the criteria which can yield an efficient cell penetration with a high degree of drug transfer. To elucidate the non-endocytic entry routes and the transduction mechanism, one possibility is to analyse interaction of TAT peptides with model membrane systems. In this study we use giant unilamellar vesicles (GUVs) as cyto-mimetic model system since the micrometer scale of the GUVs enables microscopic observation of these liposomes.<br /> In this study we applied high-speed single-particle tracking (SPT) and confocal laser scanning microscopy to systematically examine factors that affect membrane binding, mobility and penetration of fluorescence labelled TAT peptides in the GUVs with different composition.<br /> To focus onto interaction between TAT and lipids the first experiments were performed in sucrose/glucose solution with all ions excluded from the media. As a reference we first examined the mobility of fluorescent lipids within the GUV bilayer. As expected, lipid mobility varied clearly with the phase state of the membranes, whereas peptide mobility was independent on membrane hydrophobic core, but dependent on headgroup of lipids in the bilayer.<br /> CLSM experiments revealed that in GUVs formed by phosphatidylcholine (PC) and cholesterol no translocation of TAT peptides but just accumulation on the membrane. The same effect was observed also for anionic GUVs containing 15-30 mol % phosphatidylserine (PS). Additional SPT experiments and evaluation of diffusion coefficients revealed that TAT peptides “float” on neutral membranes and they are partial inserted in the headgroup of anionic bilayers. Introduction of a significant amount of anionic lipids (40 mol %) or lipids inducing locally a negative curvature into the membranes (20 mol %) affected TAT translocation across these membranes. Notably, we discovered that TAT peptides were not only able to directly penetrate such membranes in a passive manner, but they were also capable of forming physical pores, which could be passed by small but not large dye tracer molecules.<br /> For the physiological relevance of the study, additional experiments in the presence of salt solutions were performed. CLSM experiments showed that physiological salt solution dramatically changed the TAT interaction with the GUV membrane. Binding of TAT to GUVs of all employed compositions was completely lost, and the peptides now efficiently translocated into the GUV interior. In confocal images no membrane staining was observable and dye release indicated again pore formation. Also the sensitive single molecule microscope did not detect any trace of peptides on the GUV surface. This result was obtained for neutral or anionic, liquid-ordered or liquid-disordered membranes. Also, there was no difference for GUVs without cholesterol or in case of other salt solutions at the same concentration, 100 mM (CaCl₂, CaCO₃ or PBS).