Surface polyethylene glycol enhances substrate-mediated gene delivery by nonspecifically immobilized complexes

Abstract

Substrate-mediated gene delivery describes the immobilization of gene therapy vectors to a biomaterial, which enhances gene transfer by exposing adhered cells to elevated DNA concentrations within the local microenvironment. Surface chemistry has been shown to affect transfection by nonspecifically immobilized complexes using self-assembled monolayers (SAMs) of alkanethiols on gold. In this report, SAMs were again used to provide a controlled surface to investigate whether the presence of oligo(ethylene glycol) (EG) groups in a SAM could affect complex morphology and enhance transfection. EG groups were included at percentages that did not affect cell adhesion. Nonspecific complex immobilization to SAMs containing combinations of EG- and carboxylic acid-terminated alkanethiols resulted in substantially greater transfection than surfaces containing no EG groups or SAMs composed of EG groups combined with other functional groups. Enhancement in transfection levels could not be attributed to complex binding densities or release profiles. Atomic force microscopy imaging of immobilized complexes revealed that EG groups within SAMs affected complex size and appearance and could indicate the ability of these surfaces to preserve complex morphology upon binding. The ability to control the morphology of the immobilized complexes and influence transfection levels through surface chemistry could be translated to scaffolds for gene delivery in tissue engineering and diagnostic applications.

Fig. 1

Cell adhesion on EG-containing SAMs. Images were captured 48 h after seeding cells on SAMs of (A) 0% EG/100% COO⁻, (B) 20% EG/80% COO⁻, (C) 40% EG/60% COO⁻, (D) 60% EG/40% COO⁻, (E) 80% EG/20% COO⁻ and (F) 100% EG, all lacking immobilized DNA complexes. Scale bars correspond to 100 μm.

Fig. 2

DNA complex immobilization on EG-containing SAMs. The amount of immobilized radiolableled DNA was determined for SAMs with increasing percentages of EG groups in a background of MUA (COO⁻), for complexes formed at N/P of 10 (A) and 25 (B). Values are reported as the mean ± SD.

Fig. 3

EG-containing SAMs and release. Radiolabeled DNA was used to quantify the amount of DNA released from each type of SAM (• 0% EG, ■ 20% EG, ▲ 40% EG, ○ 100% EG) into serum-containing media. Complexes formed at N/P 10 (A) and 25 (B). Values are reported as cumulative percentage released, reported as the mean ± SD at each time point.

Fig. 4

EG-containing SAMs and substrate-mediated transfection. SAMs were formed with increasing percentages of EG groups in a background of MUA (COO⁻) and transfection was assayed for complexes formed at N/P of 10 (A) and 25 (B) by normalizing luciferase levels to total protein amounts. Values are reported as the mean ± SD (*p < 0.05, **p < 0.01).

Fig. 5

Cell adhesion on complexes immobilized on EG-containing SAMs. The morphology and adhesion of cells was observed 48 h after seeding cells on SAMs of (A) 100% EG with PEI concentration equivalent to N/P 10, (B) 100% EG with PEI concentration equivalent to N/P 25, (C) 100% EG with PEI–DNA complexes formed at N/P 10 and (D) 100% EG with PEI–DNA complexes formed at N/P. Scale bars correspond to 100 μm.

Fig. 6

EG-containing SAMs and transfection efficiency. SAMs were formed with increasing percentages of EG groups in a background of MUA (COO⁻) and transfection efficiency was assayed for complexes formed at N/P of 10 (A) and 25 (B) by counting the number of cell expressing β-galactosidase and dividing by the total number of cells. Values are reported as the mean ± SD (*p < 0.05, **p < 0.01).

Fig. 7

Substrate-mediated transfection on SAMs with backgrounds of EG and COO⁻. SAMs were formed with alkanethiols containing various terminal functional groups, including OH, CH₃, EG and COO⁻, in backgrounds of 40% EG (A and B) and 60% COO⁻ (C and D). Transfection was assayed for complexes formed at N/P of 10 (A and C) and 25 (B and D) by normalizing luciferase levels to total protein amounts. Values are reported as the mean ± SD (*p < 0.05, **p < 0.01).

Fig. 8

AFM images and analysis of complexes immobilized on SAMs. Complexes were formed at N/P of 10 (A–D) and N/P 25 (E–H) and immobilized on SAMs formed with 0% EG/100% COO⁻ (A, B, E, F) or 40% EG/60% COO⁻ (C, D, G, H). Pixel brightness in images (A, C, E, G) corresponds to particle height. Scale bars correspond to 1.0 μm. Height analysis histogram (B, D, F, H) reports analysis of image to left.