TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors - PubMed (original) (raw)

TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors

V P Torchilin et al. Proc Natl Acad Sci U S A. 2001.

Abstract

To achieve an efficient intracellular drug and DNA delivery, attempts were made to target microparticulate drug carriers into cytoplasm bypassing the endocytotic pathway. TAT peptides derived from the HIV-1 TAT protein facilitate intracellular delivery of proteins and small colloidal particles. We demonstrated that relatively large drug carriers, such as 200-nm liposomes, can also be delivered into cells by TAT peptide attached to the liposome surface. Liposomes were fluorescently labeled with membranotropic rhodamine-phosphatidylethanolamine or by entrapping FITC-dextran. Incubation of fluorescent TAT liposomes with mouse Lewis lung carcinoma cells, human breast tumor BT20 cells, and rat cardiac myocyte H9C2 results in intracellular localization of certain liposomes. Steric hindrances for TAT peptide x cell interaction (attachment of TAT directly to the liposome surface without spacer or the presence of a high MW polyethylene glycol 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 on the translocation of TAT liposomes into cells, confirming the energy-independent character of this process. The approach may have important implications for drug delivery directly into cell cytoplasm.

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Figures

Figure 1

Figure 1

Synthesis of pNP-PEG-PE and its interaction with amino-containing ligand.

Figure 2

Figure 2

Size distribution patterns for various liposomal preparations used in the study: LIP (A), LIP-NGPE-TAT (B), LIP-PEG(5000) (C), LIP-pNP-PEG(3000) (D).

Figure 3

Figure 3

Schematic pattern of the interaction of TAT peptide-containing liposomes with cell surface: LIP-NGPE-TAT (TAT peptide is located directly on the surface of liposome) (A), LIP-pNP-PEG(3000)-TAT (TAT peptide is coupled via PEG spacer) (B), LIP-PEG(2000)/pNP-PEG(3000)-TAT (TAT peptide is attached to PEGylated liposomes; the length of protecting PEG is smaller that the length of PEG spacer) (C), and LIP-PEG(5000)/pNP-PEG(3000)-TAT (TAT peptide is attached to PEGylated liposomes; the length of protecting PEG is bigger that the length of PEG spacer) (D). 1, phospholipid constituting liposomes; 2, PE anchor for TAT peptide or TAT peptide-bearing PEG spacer; 3, TAT peptide; 4, PEG chains of variable length; and 5, hypothetical sites of TAT peptide interaction with the cell surface.

Figure 4

Figure 4

Bright field (Left) and fluorescent (Right) microscopy of cell cultures treated with different preparations of Rh-PE labeled liposomes (37°C; ×600): H9C2 and LIP (A), BT20 and LIP-pNP-PEG(3000) (B), BT20 and LIP-NGPE-TAT (C), BT20 and LIP-pNP-PEG(3000)-TAT (D), and LLC and LIP-pNP-PEG(3000)-TAT (E). (Blue spots seen in pictures under light microscopy are because of variations in light intensity, camera capture settings, and processing.) (F and G) Images represent the results of confocal microscopy of BT20 treated with LIP-pNP-PEG(3000)-TAT at 37°C: single cell viewed from the top cell surface (F); single cell viewed at the mid-point of the cell thickness (≈0.8-μm distance) (G).

Figure 5

Figure 5

Bright field (Left) and fluorescent (Right) microscopy of cell cultures treated with Rh-PE-liposomes (37°C; ×400): BT20 and LIP-PEG(2000) (A), H9C2 and LIP-PEG(5000)/pNP-PEG(3000)-TAT (B), BT20 and LIP-PEG(5000)/pNP-PEG(3000)-TAT (C), H9C2 and LIP-PEG(2000)/pNP-PEG(3000)-TAT (D), and BT20 and LIP-PEG(2000)/pNP-PEG(3000)-TAT (E).

Figure 6

Figure 6

Bright field (Left) and fluorescent (Right) microscopy of viable cells treated with Rh-PE-liposomes in the presence of metabolic inhibitors (×400): BT20 and LIP-pNP-PEG(3000)-TAT, 4°C (A); BT20 and LIP-pNP-PEG(3000)-TAT, 0.1% sodium azide, 37°C (B); and LLC and LIP-pNP-PEG(3000)-TAT, 1% IAA, 37°C (C).

Figure 7

Figure 7

Bright field (Left) and fluorescent (Center) microscopy of H9C2 treated with free FITC-dextran and FITC-dextran-loaded Rh-PE labeled liposomes (37°C; ×400): H9C2 and free FITC-dextran (A); H9C2 and LIP-pNP-PEG(3000)-TAT/FITC-dextran, TRITC filter (B); and H9C2 and LIP-pNP-PEG(3000)-TAT/FITC-dextran, FITC filter (C). (Right) Images represent the results of confocal microscopy of BT20 treated with FITC-dextran-loaded Rh-PE liposomes (37°C; pseudocolor enhancement): single cell under FITC filter (D); single cell under Rh filter (E); and composite of superimposed layers from D and E (F).

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