Calcium condensed LABL-TAT complexes effectively target gene delivery to ICAM-1 expressing cells

Abstract

Targeted gene delivery using nonviral vectors is a highly touted scheme to reduce the potential for toxic or immunological side effects by reducing dose. In previous reports, TAT polyplexes with DNA have shown relatively poor gene delivery. The transfection efficiency has been enhanced by condensing TAT/DNA complexes to a small particle size using calcium. To explore the targetability of these condensed TAT complexes, LABL peptide targeting intercellular cell-adhesion molecule-1 (ICAM-1) was conjugated to TAT peptide using a polyethylene glycol (PEG) spacer. PEGylation reduced the transfection efficiency of TAT, but TAT complexes targeting ICAM-1 expressing cells regained much of the lost transfection efficiency. Targeted block peptides properly formulated with calcium offer promise for gene delivery to ICAM-1 expressing cells at sites of injury or inflammation.

Figure 1

HPLC chromatograms for TAT, TAT-PEG, and TAT-PEG-LABL confirmed purity > 95%.

Figure 2

Electrospray ionization (ESI) mass spectra of (A) TAT, (B) TAT-PEG, and (C) TAT-PEG-LABL were in agreement with calculated masses.

Figure 3

Gel electrophoresis of (A) TAT/DNA and (B) TAT-PEG/DNA complexes at different N/P ratios. (C) TAT-PEG-LABL/DNA complexes at an N/P ratio of 30 with different amounts of TAT-PEG-LABL combined with TAT-PEG. All complexes at all N/P ratios limited the mobility of DNA.

Figure 4

DLS was used to determine the size of TAT/DNA, TAT-PEG/DNA, 25% TAT-PEG-LABL/DNA, and 50% TAT-PEG-LABL/DNA complexes at an N/P ratio of 30 with different concentration of CaCl₂. (A) The hydrodynamic diameter of complexes were determined in deionized water and (B) in serum-free F12K media. (C) The hydrodynamic diameter of complexes (formed with 150 mM CaCl₂) in F12K media were stable over time. For missing data points, diameter was >1 µm.

Figure 5

Transmission electron micrographs of (A) TAT/DNA, (B) TAT/DNA-Ca, (C) TAT-PEG/DNA, (D) TAT-PEG/DNA-Ca, (E) 25% TAT-PEG-LABL/DNA, (F) 25% TAT-PEG-LABL/DNA-Ca (G) 50% TAT-PEG-LABL/DNA, and (H) 50% TAT-PEG-LABL/DNA-Ca complexes. Complexes were formed at an N/P ratio of 30 without CaCl₂ (left panel) or with 75 mM of CaCl₂ (right panel). Scale bars are 500 nm.

Figure 6

A heparin displacement assay for (A) TAT/DNA, (B) TAT-PEG/DNA, (C) 25% TAT-PEG-LABL/DNA, and (D) 50% TAT-PEG-LABL/DNA complexes was used to assess the effect of calcium chloride concentration (0, 30, 75, 150, 300 mM) on complex stability. Complexes were formed at an N/P of 30 and incubated for 30 min with increasing heparin concentrations (0.05–0.35 U). Free DNA is shown as a control (C) to the left.

Figure 7

TAT peptide and derivatives showed low cytotoxicity in comparison to PEI (A) in unactivated and (B) in activated A549 cells, which overexpress ICAM-1.

Figure 8

Transfection efficiencies of TAT peptide derivative/DNA complexes in A549 cells. (A) TAT/DNA and TAT-PEG/DNA complexes at an N/P ratio of 30 with different concentrations of calcium chloride (B) TAT/DNA complexes at different N/P ratios (C) TAT-PEG/DNA complexes at different N/P ratios. L= Lipofectamine.

Figure 9

Relative ICAM-1 expression level in A549 cells after activation with TNF-α for 24 hrs and 48 hrs (* = p<0.05, t-test).

Figure 10

Transfection efficiencies of TAT peptide derivative/DNA complexes in activated A549 cells (overexpressing ICAM-1) at different concentrations of calcium chloride. Complexes were formed at an N/P ratio of 30. L= Lipofectamine (* = p<0.05, one-way ANOVA, Tukey post test).

Figure 11

Transfection efficiencies of TAT/DNA and 50% TAT-PEG-LABL/DNA complexes in activated A549 cells (overexpressing ICAM-1) after incubation with free LABL peptide or anti-ICAM-1 mAb prior to exposure to TAT complexes. Complexes were formed at an N/P ratio of 30 and 150 mM CaCl₂. (* = p<0.05, ** = p<0.01, one-way ANOVA, Tukey post test).

Figure 12

Transfection efficiencies of 25% and 50% TAT-PEG-LABL/DNA complexes in normal and activated A549 cells. Complexes were formed at an N/P ratio of 30 and and 150 mM CaCl₂. (* = p<0.05, t-test)

Figure 13

Micrographs of (A) TAT-PEG/DNA complexes and (B) 50% TAT-PEG-LABL/DNA complexes in A549 cells (activated with TNF-α) after 4 hrs of incubation at 37°C. Complexes were formed at an N/P ratio of 30 and a CaCl₂ of 150 mM. (1 = DAPI fluorescence (cell nuclei), 2 = TOTO-3 fluorescence (DNA), 3 = Merged DAPI and TOTO-3 fluorescence, 4 = Merged DAPI, TOTO-3 fluorescence, and bright field transmission.)