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.

Introduction

Elastin-like polypeptides (ELP) are biologically compatible polymers that are created through a recombinant synthesis process. They are structurally based on mammalian tropo-elastin with a pentapeptide repeat unit of Valine-Proline-Glycine-X-Glycine, where X can be any amino acid except proline. One of ELP’s most studied and notable properties is its ability to undergo a lower critical solution phase transition (LCST) behavior that allows it to precipitate and form coalesced structures at elevated temperatures above its transition temperature (T_t). The ability to manipulate the T_t of ELP has been well documented and can be performed by changing any combination of polymer concentration, polymer molecular weight, polymer repeat structure, Hoffmeister anions, or pH.[1–7] A lesser studied behavior is the ability to control the size of the aggregates that form above the T_t. Previously, we showed that similar to T_t, the hydrodynamic radius (R_h) of the polymers could be systematically controlled by altering the polymer type and the solution conditions. We found that NaCl concentration and polymer type have the largest effect on R_h above the T_t, followed by polymer concentration and pH.[8]

Surface roughness is known to play an important role in promoting cellular adhesion and improving cellular viability.[16–17] For example, neuron and glia cells are known to be sensitive to micro and nano-scale roughness where micro-roughness can be used to guide cells into patterns and nano-roughness is important for promoting cellular attachment.[18] Nano-scale features are particularly important when studying neuronal cells as these features tend to trigger subcellular mechanisms in the sub-micron cellular structures such as the growth cone filopodia and basement membrane of axons.[19–20] Perhaps one of the best studied cell types with surface roughness is the osteoblast which is known to be sensitive to nano-scale roughness.[21] A study by Zareidoost et al. created eight etched titanium substrates with nanometer roughness ranging from 4.9 to 66.5 nm. They found that cellular attachment was significantly greater for surfaces with higher nano-roughness.[22]

We previously demonstrated that ELP desiccated on a silica surface could be altered to provide different sized surface features by changing drying time and polymer concentration.[23] We also showed that similar secondary structures found in ELP aggregated in solution were present throughout the desiccation of the ELP on the silica surface.[24] These observations indicate that the same processes that govern ELP’s behavior in solution to control aggregate size could be used to control its behavior on a surface to create coatings with defined features. The research presented here seeks to explore the effect of solution parameters on the creation of surface coatings using ELP and ELP-PEI deposited onto different substrates commonly used to culture cells in vitro. To this end, we tested two different polymers to form coatings, ELP and ELP-PEI, by altering the polymer concentration (0.5, 1, 1.5 mg/mL), substrate type (glass, polystyrene, tissue-culture polystyrene), and salt concentration (None, 0.2 M PBS).

Materials and Methods

ELP Expression:

ELP (MW = 17,000 g/mol; [Valine-Proline-Glycine-Valine-Glycine]₄₀) was produced from genetically modified Escherichia coli BLR (DE3) bacteria (Novagen EMD). Purification was accomplished through successive heat-cool thermo-cycling that utilizes the LCST behavior of ELP. Comprehensive accounts of this procedure are available in previously published literature.[12,14]

ELP-PEI Copolymer Synthesis:

ELP (30 mg) was reacted to branched PEI (MW = 800 g/mol from Sigma-Aldrich, St. Louis, MO) using a two-step carbodiimide reaction. The reaction was previously optimized by altering pH and reagent concentrations.[8] In the first step, the carboxylic acid on the ELP was reacted to N-hydroxysuccinimide (NHS; Sigma-Aldrich) (10:1 molar ratio of NHS:ELP) using 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC; Sigma-Aldrich) as a catalyst (10:1 molar ratio of EDC:ELP). The reaction was performed in 3 mL MES buffer (Sigma-Aldrich) at a pH of 6.2 for 15 minutes. In the second step, this ELP solution was added dropwise into a 1 mL PEI solution (10:1 molar ratio of PEI:ELP; pH = 6.8) and the reaction was allowed to proceed for 12 hours. Unreacted reagents were removed by thermo-cycling in a centrifuge and subsequent dialysis (8,000 g/mol cutoff, 1 L of DI water). The polymer concentration after dialysis was 7.5 mg/mL. A detailed account of this procedure has been previously published.[8] The reaction conversion was determined by the O-phthalaldehyde (OPA; Thermo-Fisher, Waltham, MA) assay performed according to the manufacturer’s instructions. The reaction conversion from ELP to ELP-PEI was determined to be 29% (2.175 mg/mL). The ELP-PEI was diluted with neat ELP to bring the ratio of ELP-PEI to ELP present in solution to 0.5:0.95 as this has been previously shown to be an optimal amount of charge for several cell types to form three-dimensional aggregates, without inducing negative side-effects from the excessive positive charges present in the PEI.[12–14]

Solution Preparation:

Aqueous solutions of ELP mixed with ELP-PEI were prepared by diluting a master batch of the polymers (5 mg/mL) to the desired concentrations of the polymer (0.5, 1, 1.5 mg/mL in deionized water) and phosphate buffered saline (0 and 0.2 M).

Coating Procedure:

1 mL of the desired solution was pipetted into a 35 mm cell culture dish. Three types of cell culture dishes were used: uncoated glass (MatTek, Ashland, MA), uncoated polystyrene (Corning, Corning, NY), tissue culture polystyrene (TCPS; Corning, Corning, NY). The cell culture plates were covered and placed into a Thermo Lindberg/ Blue M digital oven (Thermo Scientific, Waltham, MA) set at 50 °C for 12 hours as described before [23]. As the coatings are dried onto the surfaces the concentration of ELP at the surface increases. Because ELP’s LCST behavior is modulated by its concentration, the high surface concentration causes the T_t to shift to temperatures below 37 °C. This phenomenon is likely responsible for the stability of the ELP coatings for up to 6 weeks in cell culture media [12–14].

Atomic Force Microscopy:

A Bruker Bioscope Catalyst AFM (Billerica, MA) was used in the standard amplitude ScanAsyst® mode with a ScanAsyst-air probe at a rate of 0.5 Hz with a scan area of 10 μm x 10 μm as described before [13]. Images were analyzed using Gwyddion software version 2.55 (Czech Metrology Institute). Average roughness was calculated using the equation R_a = (1/N)sum(|r|) using measurements made at 20 points per sample.

FT-IR Spectroscopy:

To confirm the coated surfaces consisted of ELP-based coatings, attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra were collected from 600 to 4000 cm⁻¹ at a 4 cm⁻¹ resolution using a Spectrum 100 FT-IR spectrophotometer (PerkinElmer, Waltham, MA, USA).

Stability Testing:

Cell culture media warmed to 37 °C was placed into the coated cell culture plates and placed into an incubator at 37 °C (Thermo-Fisher). Simulated cell culture media changes were performed on days 3, 5, 7, 9, and 11. AFM was performed to assess the roughness using the ScanAsyst-fluid probe and fluid probe holder.

Crosslinking:

We modified the procedure described by Tejeda-Montes et al. [25], wherein hexamethyl diisocyanate (HMDI; Sigma-Aldrich) (1:1 molar ratio of HMDI to ELP) was dissolved in 1 mL anhydrous hexanes (Sigma-Aldrich) and placed into the cell culture plate. The cell culture plate was subsequently placed into a gallon-sized zip-loc bag filled with ultra-pure nitrogen gas for 12 hours. The plate was removed from the bag and washed with hexane (25 mL), followed by ethanol (25 mL), and DI-water (50 mL) to remove any unreacted HMDI. The crosslinked samples were subjected to 30 minutes of sonication in cold water (4 °C) to test the stability of the coatings.

Optical Microscopy:

Images were taken using the brightfield setting on an Olympus IX81 inverted microscope (Olympus Corp, Tokyo, Japan) equipped with a Hamamatsu digital camera.

Statistical Analysis:

Three samples were made for each of the 36-solution conditions for a total of 108 samples. Each sample was measured in three different areas. Roughness is reported as the mean ± 95% confidence interval. Statistical analysis was performed by one-way ANOVA and Games-Howell post hoc test for uneaual variances using SPSS version 24 statistical software. Differences with p ≤ 0.05 were deemed statistically significant.

Results

Figure 1 shows the average roughness (R_a) values for ELP and ELP-PEI coatings formed onto the glass, polystyrene, and TCPS substrates. The R_a values for the uncoated glass, polystyrene, and TCPS were 2.2 ± 0.3 nm, 2.8 ± 0.3 nm, and 1.4 ± 0.1 nm, respectively. Figure 1a shows the ELP coatings that produced higher R_a values. Coatings formed on polystyrene using the ELP solution with concentration of 0.5 mg/mL in PBS produced the highest R_a value at 32.2 ± 8.8 nm (p ≤ 0.05 compared to uncoated polystyrene). Four other coatings also produced significantly rougher surfaces compared to their uncoated counterparts (p ≤ 0.05); namely, the coatings formed on TCPS using the 0.5 and 1 mg/mL ELP solutions in PBS, on polystyrene using the 1 mg/mL ELP solution in PBS, and on glass using the 0.5 mg/mL ELP solution in PBS that produced the R_a values around 24 nm. The ELP solution with concentration of 1.5 mg/mL in PBS produced an R_a value of 3.4 ± 2.7 nm on glass, which was statistically indistinct from the uncoated glass (p > 0.05). The R_a values of all other ELP coatings were between 0.31 and 0.63 nm, indicating very smooth surfaces (Fig. 1b). Notably, these values fall below the non-coated control surfaces indicating that ELP smooths out the surface.

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.

Figures 1c and 1d show the R_a values of the ELP-PEI coatings. ELP-PEI coatings on glass showed sub-nanometer R_a with values between 0.22 and 0.45 nm, with one exception: the coating prepared using 1.5 mg/mL ELP-PEI solution in PBS that showed an R_a of 6.2 ± 2.3 nm. The bee-swarm plot for this sample indicates that the roughness values fall into two groups, one centered near 16 nm and another similar to that of the control surface at 2 nm (Fig. 1c). This distribution indicates that the ELP-PEI does not evenly coat the surface for this sample, and that the underlying substrate is still visible to the AFM tip. Most ELP-PEI coatings on polystyrene and TCPS also exhibited low R_a values between 0.23 to 1 nm (Fig. 1d). Two ELP-PEI coatings on polystyrene had higher R_a values: the coatings prepared using 0.5 and 1.5 mg/mL ELP-PEI solutions in water had an R_a of 1.1 ± 0.4 nm and 11.6 ± 3.4 nm, respectively (Fig. 1d). Two ELP-PEI coatings on TCPS showed higher R_a values with PBS as a solvent, wherein the 0.5 and 1 mg/mL ELP-PEI solutions in PBS had higher R_a values at 27.9 ± 5.8 nm and 1.9 ± 0.1 nm compared to those prepared in DI water that had the R_a values at 0.37 ± 0.03 nm and 0.56 ± 0.19 nm, respectively (p ≤ 0.05).

Overall, the uncoated surfaces were found to have intermediate values of surface roughness to that of the coated surfaces (Fig. 1). While the lower R_a values were expected as ELP coatings have previously been shown to decrease the Ra of TCPS [13], the current work determined that systematically altering the processing and environmental conditions produce a wide range of lower and higher R_a values for the coatings.

Optical microscopy shows that the surfaces have varied micrometer-sized morphologies that were present over the entirety of the surface. For example, for 0.5 mg/mL ELP and ELP-PEI solutions, the morphology of the coatings can be altered by changing the polymer type, solvent, and substrate type (Fig. 2). Using polystyrene as a substrate leads to slightly wavy ELP coatings (Fig. 2a) that can either be modified by changing the polymer from ELP to ELP-PEI to form smaller bumps (Fig. 2b) or by preparing the solutions in PBS to create a more complex texture (Fig. 2c). ELP and ELP-PEI formed smooth coatings on glass (Fig. 2d); however, we in general found that glass caused the polymer to dewett and form pockets of the polymer.

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.

FT-IR spectroscopy was used to confirm that the coating process resulted in an ELP-based coating and that the images collected from AFM and optical microscopy are those of an ELP coating (Fig. 3). The primary peak for ELP is present at 1650 cm⁻¹ and represents the polypeptide amide bond. The peak at 1650 cm⁻¹ is notably absent for the non-coated substrates (PS, glass) with glass having a strong Si-O band at 900 cm⁻¹ and PS showing characteristic bands at 1490 and 1450 cm⁻¹ for the C-H bonds. After coating with ELP, the substrates showed the characteristic ELP polypeptide band at 1650 cm⁻¹ indicating that ELP was successfully deposited onto the surface.

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.

Media changes were performed to test the stability of the coatings if they were to be used for cell culture. The samples were chosen to represent two of the substrates (polystyrene and TCPS), both polymer types (ELP and ELP-PEI), and the highest and lowest polymer concentrations (0.5 and 1.5 mg/mL). Glass was omitted, as it caused the polymer to dewett and would be the least likely to survive media changes. While the coatings were not removed from the substrates over the 11-day media exposure, AFM showed changes in surface architecture that occurred after every media change. The surface of the ELP-PEI coated on polystyrene substrate at a concentration of 0.5 mg/mL solution in PBS began at day 0 with a smooth appearance (Fig. 4a) that, by day seven, appeared to be covered in the regular distribution of small holes (Fig. 4c). By day 11, the small holes had turned into larger agglomerates on the surface (Fig. 4d).

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.

The R_a values obtained for the selected ELP and ELP-PEI coatings during the media exposure over 11 days are shown in Fig. 5. ELP-PEI coated on polystyrene using the 1.5 mg/mL solution in water was seen to increase in R_a from day 0 at 3.4 ± 1.3 nm to 41.4 ± 7.3 nm by day 11; ELP-PEI coated on polystyrene using the 0.5 mg/mL solution in PBS was also shown to increase in roughness from 0.29 ± 0.02 nm on day 0 to 39.1 ± 6.4 by day 11 (p ≤ 0.05). Similar results were obtained for ELP coated on TCPS using the 1.5 mg/mL solution in water where the initial roughness was low on day 0 at 0.24 ± 0.02 nm and increased to 32.3 ± 4.5 nm by day 11 (p ≤ 0.05). The ELP coated on TCPS using the 0.5 mg/mL solution in PBS had an initially high roughness on day 0 at 22.7 ± 3.5 nm that decreased to 13.6 ± 2.1 nm on day 3 (p ≤ 0.05), but increased back to 28.3 ± 8.5 nm by day 11 (p ≤ 0.05 versus day 3; p > 0.05 versus day 0).

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.

The average roughness of the coatings showed no statistical change after crosslinking with HMDI, apart from the ELP-PEI sample at 0.5 mg/mL with PBS that increased in roughness from 3.4 ± 1.3 nm (Fig. 5) to 28.3 ± 18.1 nm (Fig. 6) (p ≤ 0.05). The crosslinked samples were subjected to sonication in cold water to test their stability. There was no statistical change in R_a for the samples before and after sonication.

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).

Discussion

ELP provides the functionality that cells need to interact with the coating, ELP-PEI allows us to alter the amount of surface charge that the cells are exposed to [11–15]. We first noticed that ELP formed interesting structures when coated on fused silica by a solvent casting process above the ELP’s T_t [23]. We further investigated the polymer coating phenomena using scanning electron microscopy to determine the factors that controlled the coating process. We found that polymer concentration and time were two main factors that influenced the size of the polymer structures that formed [23]. In another study, we used FT-IR spectroscopy and peak deconvolution to determine the main secondary structures that influenced how the polymers arranged themselves on the silica [24]. It was found that type-II beta-turns were the main driving force for coating formation while water was present, but as the water evaporated the secondary structures shifted to a 3(10)-helix [24]. The work presented in this paper extends our previous research to include substrate type, salt concentration, and polymer type to investigate how each of these factors influences the way ELP forms a coating when dried above its T_t. We found that altering solution conditions allows us to control the deposition of these polymers and ultimately the surface roughness. The samples that came the closest to being completely smooth were coated on glass and polystyrene at 1 mg/mL. Samples at a concentration of 0.5 mg/mL tended to form coatings that had higher R_a values than the other two concentrations. This may be due to the small amount of polymer available to evenly coat the surface and the polymer preferentially staying with itself rather than interacting with the surface. At the higher concentrations, there may be enough polymer that, after the initial adsorbing layer forms, the polymer can then add to it evenly. The addition of PBS was the other major factor that determined how rough the surfaces were after coating. Salt is known to lower the T_t of ELP and thus make it more energetically favorable to agglomerate [6, 8]. We previously showed that the addition of salt to solution increased the hydrodynamic radius of ELP and decreased the T_t [8]. The larger, more stable aggregates that are formed in solution could contribute to the increase in roughness seen when ELP solutions in PBS are used to create the coatings.

ELP-PEI showed more coating conditions that formed with lower R_a values than ELP (Fig. 1). We previously demonstrated that ELP-PEI formed smaller aggregates in solution than ELP under the same conditions.[8] These smaller aggregates likely formed the smoother surfaces with lower R_a values. Images of the ELP and ELP-PEI coatings on polystyrene show that ELP has more broad bumps and ridges than ELP-PEI, which exhibits small bumps (Fig. 2). This may lend credence to the idea that the smaller aggregates ELP-PEI formed in solution deposit onto the substrate to form smaller features. Since the hydrodynamic radius of the ELP-PEI aggregates are smaller than ELP aggregates in solution [8], the surface areas for the individual ELP-PEI aggregates are expected to be smaller than that of the individual ELP aggregates. Given the same polymer concentration is used, a smaller aggregate size will also result in a greater number of aggregates. As the total surface area of a coating will depend on the number of aggregates and the size of the individual aggregates, it follows that a larger total surface area is expected for the ELP-PEI coating compared to the ELP coating. This means that the same amount of ELP-PEI can cover more surface area of the substrate leading to more uniform coatings.

ELP-PEI coatings have historically been used for cell culture. Previous studies used a much higher polymer concentration (5 mg/mL) than what was used in this study and did not attempt to control the deposition or roughness of the coatings [12–14]. Any changes in the surface roughness of these ELP-PEI coatings during cell culture media exposure has also not been tested. We were impressed to find that that even at the highest concentration used in this study at 1.5 mg/mL, the coatings exhibited a change in surface roughness with each media change (Fig. 5). Even more interesting was the formation of micrometer-sized aggregates on the surface at day 11 for the ELP-PEI coating on polystyrene substrate prepared using a 0.5 mg/mL solution in PBS shown in Fig. 4. One would expect the dissolution of the coating with time rather than the complete rearrangement of the surface we observed. A simple explanation is that the temperature of the media at 37 °C is higher than the T_t of the ELP and ELP-PEI used and is warm enough to maintain the coatings, but whenever the plates are removed from the incubator for a media change, the small media volume cools enough that the ELP and ELP-PEI can begin to rearrange without dissolution of the coatings.

Crosslinking ELP with HMDI has been done before to form biocompatible membranes. Tejedas-Montes et al. used a VPG[I:K]G₁₂₀ ELP dissolved in DMF and dried it onto silicon molds with different micrometer patterns. The dried ELP was crosslinked with HMDI to maintain the integrity of the membranes. The excess HMDI was removed with subsequent washing, and mesenchymal stem cells were successfully cultured atop it to demonstrate the biocompatibility of the HMDI-crosslinked coatings [25]. ELP and ELP-PEI are soluble in cold water and the added energy from sonication can remove any non-crosslinked coating from the cell culture plate. We found that crosslinking the coatings with HMDI was effective at preventing such removal as well as any change in the average roughness (Fig. 6).

Conclusions

This study demonstrates the viability of using predefined solution conditions to control how ELP and ELP-PEI deposit onto three different materials used to create cell-culture dishes. The coatings were shown to change both in average roughness and in morphology when media changes were performed. Crosslinking with HMDI prevented the coatings from rearranging and maintained their surface roughness during stability testing. Further refinement of this work could see the creation of surfaces that exhibit controlled roughness that encourage in vitro cultures of a variety of cell types.