Functionalization of reactive polymer multilayers with RGD and an antifouling motif: RGD density provides control over human corneal epithelial cell-substrate interactions

Abstract

Our study demonstrates that substrates fabricated using a "reactive" layer-by-layer approach promote well-defined cell-substrate interactions of human corneal epithelial cells. Specifically, crosslinked and amine-reactive polymer multilayers were produced by alternating "reactive" deposition of an azlactone-functionalized polymer [poly(2-vinyl-4,4-dimethylazlactone)] (PVDMA) and a primary amine-containing polymer [branched poly(ethylene imine)] (PEI). Advantages of our system include a 5- to 30-fold decrease in deposition time compared to traditional polyelectrolyte films and direct modification of the films with peptides. Our films react with mixtures of an adhesion-promoting peptide containing Arg-Gly-Asp (RGD) and the small molecule D-glucamine, a chemical motif which is nonfouling. Resulting surfaces prevent protein adsorption and promote cell attachment through specific peptide interactions. The specificity of cell attachment via immobilized RGD sequences was verified using both a scrambled RDG peptide control as well as soluble-RGD competitive assays. Films were functionalized with monotonically increasing surface densities of RGD which resulted in both increased cell attachment and the promotion of a tri-phasic proliferative response of a human corneal epithelial cell line (hTCEpi). The ability to treat PEI/PVDMA films with peptides for controlled cell-substrate interactions enables the use of these films in a wide range of biological applications.

Figure 1

A schematic demonstrating functionalization of PEI/PVDMA multilayer films with mixed solutions of RGD and d-glucamine. Films were allowed to react with solutions (total concentration of 110 mM) that ranged from 0% RGD (100% d-glucamine) to 20% RGD (80% d-glucamine).

Figure 2

After 24 hours in culture, hTCEpi cells did not attach significantly to substrates fabricated with 0, and 1% RGD solutions (a–b). However, increased attachment was noted on substrates functionalized with solutions containing 3–20% RGD/d-glucamine (c–h). The cells were stained with SYTO-11 green fluorescent nucleic acid stain.

Figure 3

hTCEpi cells demonstrated significant increase in attachment to films functionalized with 5% RGD/d-glucamine solutions compared to 0 to 3% RGD/d-glucamine solutions. As the percentage of RGD in the reactive solution increases from 5 to 20%RGD/d-glucamine, the number of attached cells per 10X image also increases. (*0.01 ≤ P < 0.05, **0.001 ≤ P < 0.01, ***P < 0.001 compared to 0% RGD control)

Figure 4

Covalent multilayer films were functionalized with either solutions containing RGD or the scrambled negative control peptide, RDG. hTCEpi cells seeded onto RDG surfaces (a–g) were unable to attach. hTCEpi cells seeded onto RGD surfaces (h–n) attached to spots fabricated from 3 to 20% RGD/d-glucamine solutions. Cells were allowed to attach for 24 hours and were then stained with SYTO-11 green fluorescent nucleic acid stain.Scale bar equals 1 mm.

Figure 5

hTCEpi cells detached from PEI/PVDMA surfaces treated with RGD/d-glucamine when exposed to soluble RGD. Soluble RGD was added to the medium 24 hours after cell seeding onto surfaces. In the presence of 0.1 and 1 mM soluble RGD, hTCEpi cells detached from surfaces treated with 3 and 5% RGD/d-glucamine solutions (b and c) within 4 hours. Cells on surfaces fabricated from 7 to 20% RGD/d-glucamine solutions (d–g) demonstrated little to no detachment in the presence of 0.1 mM soluble RGD. However, in the presence of 1 mM soluble RGD, hTCEpi cells on 7 to 15% RGD/d-glucamine surfaces (d–f) demonstrated surface RGD-density dependent detachment. Cells on TCPS (a) and substrates fabricated from 20% RGD solutions (g) did not demonstrate detachment in the presence of 0.1 and 1 mM soluble RGD. Each graph is representative of the remaining cells attached to TCPS or a given %RGD/d-glucamine-modified surface. Symbols: ○- no peptide added to culture medium; △-1 mM RDG added to culture medium; ●- 0.1 mM RGD added to culture medium;▲- 1 mM RGD added to culture medium. Error bars denote the standard error of the mean. Graphs are representative of experiments performed in triplicate.

Figure 6

hTCEpi cells demonstrated proliferation rates dependent on the surface density of RGD. On 0 to 3% RGD/d-glucamine films, cells did not attach well. Those cells that initially attached to the surface ultimately detached over the course of 5 days. Cells on surfaces fabricated with 5% RGD/d-glucamine did not proliferate, while cells on surfaces fabricated with 7% RGD/d-glucamine showed a slight increase in cell numbers. The highest rates of proliferation were observed on surfaces fabricated from 9–20% RGD/d-glucamine solutions. (*0.01 ≤ P < 0.05, **0.001 ≤ P < 0.01, ***P < 0.001 compared to 0, 1, 3, and 5 % RGD from day 5)