Tuning the mechanical properties of bioreducible multilayer films for improved cell adhesion and transfection activity

Abstract

A simple approach to the mechanical modulation of layer-by-layer (LbL) films is through manipulation of the film assembly. Here, we report results based on altering the salt concentration during film assembly and its effect on film rigidity. Based on changes in film rigidity, cell adhesion characteristics and transfection activity were investigated in vitro. LbL films consisting of reducible hyperbranched poly(amide amine) (RHB) have been implemented along with DNA for investigating fibroblast adhesion on [RHB/DNA](n/2) films with varying rigidities. The rigidity was varied by changing the ionic concentration of the deposition solution between 0.01 m NaCl and 1.0 m NaCl. Molecular force probe (MFP) measurements were performed to measure the apparent Young's modulus, E(APP), of the films in situ. Cell adhesion and stress-fiber characteristics were investigated using total internal reflectance microscopy (TIRF-M). The average cell peripheral area, fiber density and average fiber length during 5 days of cell growth on films with either low (below 2.0 MPa) or high (above 2.0 MPa) film elastic modulus were investigated. Transfection studies were performed using gfpDNA and SEAP-DNA to investigate if changes in cell adhesion affect transfection activity. Furthermore, cell proliferation and cytotoxicity studies were used to investigate cellular viability over a week. The results have shown that surface modification of bioreducible LbL films of controlled thickness and roughness promotes cellular adhesion, stress-fiber growth and increased transfection activity without the need for an additional adhesive protein pre-coating of the surface or chemical cross-linking of the film.

Figure 1

Thickness data obtained from ellipsometry and AFM roughness (R_a) data of films with increasing number of (RHB/DNA) layers and increasing ionic concentration of the deposition solution.

Figure 2

AFM height images of (RHB/DNA)_n/2 films, 10μm × 10μm. In the left column the films were deposited with 0.01 M NaCl and the images in the right column were deposited with 0.5 M NaCl. a,b) (RHB/DNA)₆, c,d) (RHB/DNA)₁₂, e,f) (RHB/DNA)₁₆, and g,h) (RHB/DNA)₂₀. All images were scanned with Z = 200 nm.

Figure 3

Cell proliferation studies using LDH reagent to quantify cell proliferation and WST-1 reagent for cell viability studies. High- and low-E_APP (RHB/DNA)_n/2 films were investigated over 7 days. Cell growth on high-E_APP ((RHB/DNA)₁₆ assembled in 0.01M NaCl) over 5 days a) 1 day, b) 3 days, c) 4 days, d) 5 days e) and low-E_APP ((RHB/DNA)₁₆ assembled in 1.0M NaCl) on the same days.

Figure 4

Quantified TIRF analysis showing the average cell are (μm²/cm²) (top), average stress fiber density (middle) and number of stress-fibers (bottom) of the spreading cells for both high- and low-E_APP films calculated over 5 days.

Figure 5

Cell transfection studies with (RHB/gfpDNA)_n/2 on both high- and low-E_APP films over 5 days. Changes after day 5 were negligible and are therefore not included.

Figure 6

Transfection activity over 7 days using SEAP DNA. (RHB/DNA)_n/2 films were deposited in either 0.01 M NaCl, 0.10 M NaCl, 0.50 M NaCl, or 1.0 M NaCl and were categorized as either high- or low-E_APP films.