Ogris M, Steinlein P, Carotta S, Brunner S and Wagner E DNA/polyethylenimine transfection particles: Influence of ligands, polymer size, and PEGylation on internalization and gene expression AAPS PharmSci 2001; 3 (3) article 21 (https://www.pharmsci.org/scientificjournals/pharmsci/journal/01_21.html).

Figures and Tables

Figure 1.Tf-mediated internalization of DNA/transferrin (Tf)- polyethylenimine (PEI)800 complexes into K562 cells. K562 cells (2 x 10⁵ cells in 0.2 mL medium) were incubated for 30 minutes at either 37°C or 4°C with (A) YOYO-1 labeled DNA/PEI₈₀₀ or (B) DNA/Tf-PEI₈₀₀ complexes (N/P 4.8) generated in HBG (1.5 µg DNA in 75 µL) and, after replacing the medium, for another 30 minutes. Harvesting, staining with TOTO-3, and analyzing cells were carried out as described in the Materials and Methods section of this article. Competitive inhibition was performed in the presence of 5 mg/mL Tf during the first 30 minutes of incubation (C: PEI₈₀₀ , D: Tf-PEI₈₀₀ ). Density plots of cells incubated at 37°C are shown. The r value (uptake ratio) shown was obtained by dividing the median value of FL1/FL4 ratios obtained at 37°C by the value obtained at 4°C (no internalization).

Table 1. Specificity of Transfection

HBG indicates HEPES buffered glucose (20 mM HEPES pH 7.4, 5% glucose wt/vol); HBS, HEPES buffered saline (20 mM HEPES pH 7.4, 150 mM NaCl); PEI, polyethylenimine; RLU, relative light units Tf, transferrin.

5 x 10⁵ K562 cells in 0.5 mL medium were transfected for 4 hours with the indicated complex (containing 5 µg DNA) mixed in either HBG or HBS (N/P 6) in the presence or absence of 5 mg/mL Tf. Twenty-four hours after transfection, cells were harvested and assayed for luciferase expression as described. The data in brackets indicate the size range of the particles used. The complex size was measured as described in Blessing et al¹⁶ .

Figure 2.Influence of complex size on internalization of K562 cells. K562 cells were incubated as described in Figure 1 ; complexes (DNA/Tf-PEI₈₀₀ ) were generated at an N/P ratio of 4.8 either in HBS (large complexes) or HBG (small complexes). (A) Histogram plots for FL1 (total associated DNA, left graph) and FL4 (surface-bound DNA, right graph) of cells incubated with the indicated complexes at 37°C. Dotted line indicates mock-treated cells; bold line, cells incubated with complexes generated in HBS; narrow line, cells incubated with complexes generated in HBG. (B) FL1/FL4 uptake ratios of cells incubated at either 37°C (bold line) or 4°C (narrow line) with complexes generated in HBG (left) or HBS (right), the r value is shown for the individual graphs. (C) Laser scanning microscopy of K562 cells incubated with YOYO-1 labeled DNA/Tf-PEI₈₀₀ complexes. K562 cells were incubated with DNA/Tf-PEI₈₀₀ complexes in either HBG (left) or HBS (right) for 30 minutes at 37°C. After washing, cells were fixed and viewed with a laser scanning microscope as described in the Materials and Methods section. Central sections of the cells are shown. Green indicates YOYO-1 labeled DNA complexes; red, DiD fluorescence.

Figure 3.Electron microscopy of DNA/(ligand)-PEI₂₅ complexes. Two micrograms of DNA in 50 µL were complexed with the following amounts of (ligand)-PEI₂₅ (N/P 6) in 50 µL as described in the Materials and Methods section: (A) 1.5 µg PEI₂₅ ; (B) 1.5 µg Tf-PEI₂₅ ; (C) 1 µg PEI₂₅ + 0.5 µg Tf-PEI₂₅ ; and (D) 1.5 µg EGF-PEI₂₅ . The bar represents 200 nm in A, C, and D; 500 nm in B.

Figure 4.Cell binding, internalization, and gene transfer with Tf-PEI₂₅ 2 x 10⁵ K562 cells in 0.2 mL medium were incubated with DNA/PEI₂₅ /Tf-PEI₂₅ complexes generated in HBG (1.5 µg YOYO-1 labeled DNA complexed with total 1.125 µg (Tf)-PEI₂₅ , N/P 6.0) for 30 minutes and, after replacing the medium, for another 30 minutes. PEI₂₅ , Tf-PEI₂₅ , or a mixture of PEI₂₅ and Tf-PEI₂₅ (113 ng Tf-PEI₂₅ + 1.013 µg PEI₂₅ for 1/10, 56 ng Tf-PEI₂₅ + 1.069 µg PEI₂₅ for 1/20) were used for DNA complexation. Cells were incubated at either 37°C or 4°C. Staining with TOTO-3 and analysis were carried out as described in the Materials and Methods section. (1A) Total cellular association at 37°C or (B) uptake ratios (r) are shown; n = 4 ± SD. (C) For transfection studies, 5 ´ 104 K562 cells in 50 µL medium were transfected with the same complex formulation (containing 0.5 µg DNA and in total 375 ng [Tf]-PEI₂₅ ). Media was replaced after 30 minutes and luciferase gene expression measured after 24 hours as described; n = 2 ± SD. Data from a representative experiment are shown.

Figure 5.Antibody-mediated binding and internalization on Jurkat cells. 2 ± 10⁵ Jurkat cells in 0.2 mL medium were incubated for 30 minutes with complexes generated in HBG (N/P 6.0; 1.5 µg YOYO-1 labeled DNA in 75 µL) and, after replacing the medium, for another 30 minutes. Incubations were carried out with DNA complexed to (B) PEI₂₅ , (C) antiCD3-PEI₂₅ , (D) PEI₂₅ /antiCD3-PEI₂₅ 3/1 wt/wt, or (E) PEI₂₅ /antiCD3-PEI₂₅ 20/1 wt/wt. Control cells (no complexes) are shown in 5A. Staining with TOTO-3 and analysis was carried out as described in the Materials and Methods section. Uptake ratios (r) for each condition are shown in the individual density plots.

Figure 6.EGF-mediated internalization into KB cells. 2 x 10⁵ KB cells in 0.5 mL medium were incubated for 30 minutes with DNA/PEI₂₅ or DNA/EGF-PEI₂₅ complexes (N/P 6.0) generated in HBG (3 µg YOYO-1 labeled DNA in 150 µL) and, after replacing the medium, for another 30 minutes. Competitive inhibition was carried out with 100-fold molar excess of EGF during the first half-hour of incubation. (A) Total cellular association (FL1, YOYO-1 fluorescence). (B) Uptake ratio (r) ; n = 2-5 ± SD.

Figure 7.Influence of PEG on Tf-mediated binding and internalization on K562 cells. 2 x 10⁵ K562 cells were incubated in 0.2 mL medium with 75 µL complex generated in HBG (N/P 6.0, 1.5 µg YOYO-1 labeled DNA) as described in Figure 1. (A) Complexes used have been PEGylated with the indicated wt/wt ratios of PEG/PEI as described in the Materials and Methods section. The uptake ratio (37°C versus 4°C) was calculated as described and plotted on the graph. Full bars indicate DNA/Tf-PEI₈₀₀ ; blank bars, DNA/PEI₈₀₀ . n = 2 ± SD. (B) K562 cells were incubated with DNA/Tf-PEI₈₀₀ complexes as described in (A). Total cellular association measured in FL1 is shown. Dotted line indicates control cells; bold line, cells incubated with DNA/Tf-PEI₈₀₀ or DNA/Tf-PEI₈₀₀ /PEG; narrow line, PEG/PEI 30/1 wt/wt.

Figure 8.Influence of PEG on complex binding and internalization on KB cells. 2 x 10⁵ KB cells in 0.5 mL medium were incubated with 150 µL (N/P 6.0, 3 µg YOYO-1 labeled DNA) complex: DNA/PEI₂₅ , DNA/PEI₂₅ /Tf-PEI₂₅ = 20/1 wt/wt or DNA/EGF-PEI₂₅ . After 30 minutes, the medium was replaced, and cells were incubated for another 30 minutes. Afterward, cells were harvested, stained with TOTO-3, and analyzed as described in the Materials and Methods section. (A) Uptake ratios for the indicated complexes, either non-PEGylated (full bars) or PEGylated with a PEG/PEI wt/wt ratio of 30/1 (clear bars) are shown; n = 2 ± SD. (B-D) Total cellular association measured in FL1 (YOYO-1) is shown. Dotted line indicates control cells; bold line, cells incubated with non-PEGylated complexes; narrow line, PEGylated complexes PEG/PEI 30/1 wt/wt. (B) DNA/PEI₂₅ . (C) DNA/PEI₂₅ /Tf-PEI₂₅ . (D) DNA/EGF-PEI₂₅ .