Engineering surfaces for substrate-mediated gene delivery using recombinant proteins

Abstract

Immobilized fibronectin and other natural proteins have been utilized to enhance substrate-mediated gene delivery, with apparent contributions from the intrinsic bioactivity and also physical properties of the immobilized proteins. In this report, we investigated the use of recombinant proteins, compared to the full-length fibronectin protein, as surface coatings for gene delivery to investigate the mechanisms by which fibronectin enhances gene transfer. The recombinant fibronectin fragment FNIII(7-10) (FNIII) contains the alpha(5)beta(1) binding domain of fibronectin and supports cell adhesion, whereas the recombinant protein polymer PP-12 is also negatively charged and has a molecular weight similar to FNIII, but lacks cell binding domains. Transfection was compared on surfaces modified with FNIII, full-length fibronectin, or PP-12. The full-length fibronectin provided the greatest extent of transgene expression relative to FNIII or PP-12, which was consistent with the amount of DNA that associated with cells. FNIII had 4.2-fold or 4.7-fold lower expression levels relative to fibronectin for polyplexes and lipoplexes, respectively. PP-12 produced expression levels that were 317-fold and 12.0-fold less than fibronectin for polyplexes and lipoplexes, respectively. Although expression was greater on FNIII relative to PP-12, the levels of DNA associated per cell with FNIII were similar to or less than those with PP-12, suggesting that the bioactive sequences may contribute to an enhanced intracellular trafficking. For lipoplexes delivered on FNIII, the efficiency of intracellular trafficking and levels of caveolar DNA were greater than that observed with either the full-length fibronectin or PP-12. For polyplexes, fibronectin fragment resulted in greater intracellular trafficking efficiency compared to PP-12 protein polymer. Recombinant proteins can be employed in place of full-length extracellular matrix proteins for substrate-mediated gene delivery, and bioactive sequences can influence one or more steps in the gene delivery process to maximize transfection.

Figure 1

Transfection of NIH/3T3 cells on protein-coated substrates. Luciferase expression by NIH/3T3 cells cultured on polystyrene with adsorbed fibronectin (black bars), fibronectin fragment FNIII_7–10 (gray bars), and protein polymer (hashed bars) is shown as a function of delivery vector (PEI = polyethyleneimine, LF = Lipofectamine). Luciferase levels are reported as relative light units (RLU) normalized by total cellular protein in the sample well. Data is presented as an average of triplicate measurements ± standard deviation of the mean. A statistically significant difference (p<0.05) is denoted with different letters within the same vector condition.

Figure 2

Phase and fluorescence microscopy images of transfected cells. Representative images of NIH/3T3 cells transfected with PEI/DNA complexes immobilized to protein polymer (A,D), fibronectin fragment (B,E) and fibronectin (C,F) are shown. Images were captured with a 20x objective. Cells expressing enhanced green fluorescent protein (EGFP) are shown in green. Scale bar indicates 25 µm.

Figure 3

Phase microscopy and fluorescence microscopy images of transfected cells. Representative images of NIH/3T3 cells transfected with Lipofectamine/DNA complexes immobilized to protein polymer (A,D), fibronectin fragment (B,E) and fibronectin (C,F) are shown. Images were captured with a 20x objective. Cells expressing enhanced green fluorescent protein (EGFP) are shown in green. Scale bar indicates 25 µm.

Figure 4

DNA immobilization to the substrate. The amount of DNA immobilized to polystyrene with adsorbed fibronectin (black bars), fibronectin fragment FNIII_7–10 (gray bars), and protein polymer (hashed bars) was measured as a function of delivery vector. Data is presented as an average of triplicate measurements ± standard deviation of the mean. A statistically significant difference (p<0.05) is denoted with different letters within the same vector condition.

Figure 5

Plasmid association to cells. NIH/3T3 cells were transfected by surface delivery from polystyrene coated with fibronectin (black bars), fibronectin fragment FNIII_7–10 (gray bars), and protein polymer (hashed bars). The number of plasmid copies associated per cell is shown as a function of delivery vector. Data is presented as an average of triplicate measurements ± standard deviation of the mean. A statistically significant difference (p<0.05) is denoted with different letters within the same vector condition.

Figure 6

Efficiency of intracellular trafficking. NIH/3T3 cells were transfected by surface delivery from polystyrene coated with fibronectin (black bars), fibronectin fragment FNIII_7–10 (gray bars), and protein polymer (hashed bars). Luciferase expression is normalized by the amount of cell-associated DNA and presented as a function of protein coating and vector. Data is presented as an average of triplicate measurements ± standard deviation of the mean. A statistically significant difference (p<0.05) is denoted with different letters within the same vector condition.

Figure 7

Co-localization of Cy5-labeled plasmid with caveolae within NIH/3T3 cells was quantified using confocal microscopy. Blue indicates nuclear and cytoplasmic nucleic acids, red indicates Cy5-labeled plasmid, and green indicates caveolae (A). Red arrows indicate Cy5-labeled DNA, green arrows indicate caveolae, and white arrows indicate co-localized Cy5-labeled DNA and caveolae. Magnified section of A is presented, as indicated by outline (B). Scale bars indicate 15 µm. Confocal images above depict transfected cells with Lipofectamine on PP-12 protein polymer. NIH/3T3 cells were transfected by surface delivery from polystyrene coated with fibronectin (black bars), fibronectin fragment FNIII_7–10 (gray bars), and protein polymer (hashed bars), and caveolar DNA was calculated as a percentage of cellular DNA (C). Data is presented as an average of triplicate measurements ± standard deviation of the mean. A statistically significant difference (p<0.05) is denoted with different letters within the same vector condition.