Manipulating the solution environment to control the surface roughness of elastin-based polymer coatings

Abstract

Elastin-like polypeptides (ELP) have been used as a genetically-engineered, biocompatible substitute for elastin. Cell culture coatings prepared using ELP conjugated to low molecular weight polyethyleneimine (PEI) entices cells to form three-dimensional cellular aggregates that mimic their in vivo counterparts. This study seeks to control the deposition of the ELP and ELP-PEI molecules to control the roughness of the final coatings. The two polymers were coated onto three different substrates (glass, polystyrene, tissue-culture polystyrene) and the solution environment was altered by changing the polymer concentration (0.5, 1.0, 1.5 mg/mL) and/or salt concentration (None, 0.2 M phosphate buffered saline) for a total of 36 conditions. Atomic force microscopy (AFM) was used to measure the average roughness (R_a) of the samples and found that ELP coated samples had a higher R_a than their ELP-PEI counterparts. The coatings were tested for stability by performing cell culture media changes every three days for 11 days. AFM showed that the average roughness of the tested samples increased with each media change. To address this, the surfaces were crosslinked using hexamethyl diisocyanate, which minimized the change in surface roughness even when subjected to an intense sonication process. This study provides parameters to achieve elastin-based coatings with controlled roughness that can be used to support stable, long-term in vitro cell culture.

Keywords: Elastin-like polypeptide; atomic force microscopy; coating; roughness.

Figure 1.

Average roughness (R_a) for (a,b) ELP and (c,d) ELP-PEI coatings formed onto three different substrates (glass, PS, TCPS) by changing the polymer concentration (0.5, 1.0, 1.5 mg/mL) and/or salt concentration (Water, PBS). Results are reported as the mean ± 95 % confidence interval. * indicates statistically significant difference (p ≤ 0.05) versus the respective uncoated substrate.

Figure 2.

Optical microscopy images of ELP and ELP-PEI coatings prepared using 0.5 mg/mL solutions show uniform micrometer level features that can be altered by the polymer type, solvent, and substrate type.

Figure 3.

FT-IR spectra of the coated and uncoated samples demonstrating the successful application of ELP onto the base substrates. The dashed red line indicates the peak at 1650 cm⁻¹ which is a characteristic polypeptide band for ELP.

Figure 4.

AFM images of coatings during stability testing. AFM images of the ELP-PEI coating on polystyrene substrate prepared using a 0.5 mg/mL solution in PBS from day 0 to day 11. The coating goes from being smooth on day 0 to forming larger aggregates by day 11.

Figure 5.

Average roughness during coating stability testing. R_a values for stability testing of coatings from simulated media changes showed that the roughness for the samples changed over time from day 0 to day 11. Results are reported as the mean ± 95 % confidence interval. * indicates statistically significant difference (p ≤ 0.05) versus day 0.

Figure 6.

Crosslinked coating average roughness before and after sonication. Results are reported as the mean ± 95 % confidence interval. The average roughness showed no statistical change after sonication (p > 0.05).