Abstract
BackgroundGene delivery approaches serve as a platform to modify gene expression of a cell population with applications including functional genomics, tissue engineering, and gene therapy. The delivery of exogenous genetic material via nonviral vectors has proven to be less toxic and to cause less of an immune response in comparison to viral vectors, but with decreased efficiency of gene transfer. Attempts have been made to improve nonviral gene transfer efficiency by modifying physicochemical properties of gene delivery vectors as well as developing new delivery techniques. In order to further improve and understand nonviral gene delivery, our approach focuses on the cell-material interface, since materials are known to modulate cell behavior, potentially rendering cells more responsive to nonviral gene transfer. In this study, self-assembled monolayers of alkanethiols on gold were employed as model biomaterial interfaces with varying surface chemistries. NIH/3T3 mouse fibroblasts were seeded on the modified surfaces and transfected using either lipid- or polymer- based complexing agents.ResultsTransfection was increased in cells on charged hydrophilic surfaces presenting carboxylic acid terminal functional groups, while cells on uncharged hydrophobic surfaces presenting methyl terminations demonstrated reduced transfection for both complexing agents. Surface–induced cellular characteristics that were hypothesized to affect nonviral gene transfer were subsequently investigated. Cells on charged hydrophilic surfaces presented higher cell densities, more cell spreading, more cells with ellipsoid morphologies, and increased quantities of focal adhesions and cytoskeleton features within cells, in contrast to cell on uncharged hydrophobic surfaces, and these cell behaviors were subsequently correlated to transfection characteristics.ConclusionsExtracellular influences on nonviral gene delivery were investigated by evaluating the upregulation and downregulation of transgene expression as a function of the cell behaviors induced by changes in the cells’ microenvronments. This study demonstrates that simple surface modifications can lead to changes in the efficiency of nonviral gene delivery. In addition, statistically significant differences in various surface-induced cell characteristics were statistically correlated to transfection trends in fibroblasts using both lipid and polymer mediated DNA delivery approaches. The correlations between the evaluated complexing agents and cell behaviors (cell density, spreading, shape, cytoskeleton, focal adhesions, and viability) suggest that polymer-mediated transfection is correlated to cell morphological traits while lipid-mediated transfection correlates to proliferative characteristics.
Highlights
Gene delivery approaches serve as a platform to modify gene expression of a cell population with applications including functional genomics, tissue engineering, and gene therapy
Transfection To examine the influence of surface chemistries described above on nonviral gene delivery, NIH/3T3 cells were seeded on Self-assembled monolayers (SAMs) modified surfaces, nonviral DNA complexes were delivered 18 h later, and transfection profiles were acquired 48 h following complex delivery
Transfection was assayed through a combination of luciferase and Pierce bicinchoninic acid (BCA) assays, and transfection profiles were acquired by dividing the relative light units (RLU) of luciferase luminescence by the total mass of protein for each sample, which normalizes the degree of luciferase expression across the entire cell population for a sample
Summary
Gene delivery approaches serve as a platform to modify gene expression of a cell population with applications including functional genomics, tissue engineering, and gene therapy. In contrast to viral-based gene delivery systems, nonviral vectors are more suitable for therapy given their lower toxicity and immune response, but are currently too inefficient to be considered relevant therapeutics [3,4,5,6,7]. This inefficiency is attributed to a number of extracellular and intracellular barriers that limit nonviral gene delivery. Intracellular processes such as endosomal escape, dissociation of the plasmid DNA from the chemical complexation vector, and nuclear entry of plasmid DNA limit transfection [8,9,10]
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