Engineering Substrate-Mediated Gene Delivery with Self-Assembled Monolayers and Soft LithographyPublic Deposited
Substrate-mediated delivery involves the immobilization of DNA, complexed with nonviral vectors, to a biomaterial or surface that supports cell adhesion. Cells cultured on the substrate are exposed to elevated DNA concentrations within the local microenvironment, which enhances transfection. As surface properties are critical to this delivery approach, self-assembled monolayers (SAMs) of alkanethiols on gold were used to investigate the effect of surface chemistries on substrate-mediated delivery. Surface hydrophilicity and ionization affected nonspecific complex immobilization and transfection, with SAMs presenting carboxylic acid groups resulting in the greatest immobilization and transfection. Subsequent studies used SAMs to investigate the effect of surfaces presenting oligo(ethylene glycol) (EG) groups on substrate-mediated delivery. 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 EG groups combined with other functional groups. Transfection enhancement could not be attributed to binding or release profiles. Atomic force microscopy imaging of immobilized complexes revealed that EG groups within SAMs affected complex size and appearance, indicating the ability of these surfaces to preserve complex morphology upon binding. To control binding and release profiles, complexes were covalently linked to SAMs presenting appropriate functional groups. Covalent tethering by multiple crosslinkers resulted in lower complex binding than corresponding conditions without the crosslinker, and no transfection. The principles guiding complex immobilization and tethering could be extended to biomaterial surfaces for tissue engineering applications. Finally, soft lithography techniques were used to pattern complex deposition and transfection, on SAMs and cell culture surfaces, for the formation of a transfected cell array, a high-throughput technique to correlate gene expression with functional cell responses. We developed an array that combines a two-plasmid system and dual bioluminescence imaging to quantitatively normalize for variability in transfection and increase sensitivity. The array was applied to quantify estrogen receptor &#945; (ER&#945;) activity in breast cancer cells. ER induction mimicked results obtained through traditional assay methods. Furthermore, the array captured a dose response to estrogen, demonstrating the sensitivity of bioluminescence quantification. Our system should serve as a standard for fabrication of transfected cell arrays to report on signaling pathways.