TAT peptide-modified liposomes for intracellular delivery of drugs and DNA.

Intracellular delivery of drugs and DNA is hampered by the cytotoxicity of some carrier systems (such as cationic lipids) and degradation of drugs or DNA as a results of endocytic processing. It has been shown that the coupling of HIV-1 TAT protein, or shorter TAT peptides derived from this protein, to other proteins and small colloidal particles facilitates their efficient intracellular delivery [1] by direct translocation through the cell membrane, bypassing thus the endocytic pathway. Recently, we have demonstrated that it is possible to attach a large number of TAT peptide molecules (up to several hundreds) to the surface of a single liposome utilizing the new coupling method developed by us and involving the use of p-nitrophenylcarbonyl-activated polyethylene glycol-phosphatidyl ethanolamine (pNP-PEG-PE) [2]. This new amphiphilic PEG derivative readily incorporates into liposomes and micelles via its PE residue, and easily binds primary amino group-containing ligands via its water-exposed pNP groups, forming stable and non-toxic urethane (carbamate) bonds. The reaction between the pNP group and the ligand amino group proceeds easily and quantitatively at pH around 8.0, and remaining free pNP groups are promptly eliminated by spontaneous hydrolysis. Therefore, pNP-PEG-PE could serve as a very convenient tool for protein and peptide attachment to the distal ends of liposome-grafted or micelle-incorporated PEG chains. To achieve and efficient intracellular delivery of drugs and DNA by targeting microparticulate drug carriers directly into the cytoplasm bypassing the endocytotic pathway, we have used pNP-PEG-PE-based technology to prepare TAT peptide-bearing liposomes. We demonstrated that relatively large drug carriers, such as 200 nm liposomes, could also be delivered into cells by TAT peptide attached to the liposome surface [3]. The incubation of fluorescently labeled rhodamine-PE(Rh-PE)-containing TAT liposomes with mouse Lewis lung carcinoma cells, human breast tumor BT20 cells, and rat cardiac myocytes H9C2 results in intracellular localization of certain liposomes. Steric hindrances for TAT peptide:cell interaction (attachment of TAT directly to the liposome surface without spacer or the presence of a high MW PEG on the liposome surface) abolish liposome internalization evidencing the importance of direct contact of TAT peptide with the cell surface. Low temperature or metabolic inhibitors, sodium azide or iodoacetamide, have little influence upon the translocation of TAT-liposomes into cells, confirming the energy-independent character of this process.