The role of surface chemistry-induced cell characteristics on nonviral gene delivery to mouse fibroblasts

Abstract

Background: 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. 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.

Results: Transfection 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.

Conclusions: Extracellular 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.

Figure 1

Normalized transfection profile for cells plated on surfaces with defined surface chemistries transfected with (a) Lipofectamine 2000 and (b) PEI. NIH/3T3 cells were seeded on SAM modified gold and tissue culture polystyrene control surfaces, nonviral DNA complexes with DNA encoding for EGFP and LUC were delivered 18 h later, and transfection profiles were acquired 48 h following complex delivery by quantifying the luciferase expression and normalizing these values per total protein amount present on the evaluated surfaces. Data is reported as mean +/− standard error of the mean of transfection profile values reported in relative light units (RLU) per mg protein. (*p < 0.05, **p < 0.01).

Figure 2

Cell viability and proliferation measured with a MTT assay on surfaces with defined surface chemistries transfected with (a) Lipofectamine 2000 and (b) PEI. The resulting absorbance was read at λ = 570 nm and results are reported as mean +/− standard error of the mean. The slope of the lines connecting each set of consecutive timepoints is indicative of the rates of cell proliferation as evidenced by the rate equation ((Δ absorbance/surface area)/Δ time).

Figure 3

(a) Cell density (b) cell spreading, and (c) cell shape index for cells plated on surfaces with defined surface chemistries. Cell density was determined by counting the number of cells per image area, cell spreading was determined by measuring the total cell area per the amount of total cells per image area, and the cell shape index was determined by the equation S = 4πA/P², where A is cell area, P is cell perimeter, and S is the cell shape factor. Data is reported as mean +/− standard error of the mean (*p < 0.05, **p < 0.01).

Figure 4

Confocal microscopy images of NIH/3T3 fibroblasts stained with TRITC phalloidin for actin filaments (red) and nuclei counterstained with DAPI (blue) on (a) -COO^-terminated, (b) -CH₃terminated, and (c) PS surfaces. Scale bar = 50 μm. Quantitative image analysis of (d) cytoskeletal stress bundles and (e) focal adhesions of NIH/3T3 fibroblasts on different biomaterial surfaces. Actin stress bundles were quantified by counting bundled actin fibers identified as bright regions of the TRITC stain, while focal adhesions were quantified by counting the amount of lamellipodial protrusions, which have previously been shown to directly correlate to focal adhesion abundance. Data is reported as mean +/− standard error of the mean (*p < 0.05, ***p < 0.001).