Avidin Adsorption to Silk Fibroin Films as a Facile Method for Functionalization

Abstract

Silk fibroin biomaterials are highly versatile in terms of materials formation and functionalization, with applications in tissue engineering and drug delivery, but necessitate modifications for optimized biological activity. Herein, a facile, avidin-based technique is developed to noncovalently functionalize silk materials with bioactive molecules. The ability to adsorb avidin to silk surfaces and subsequently couple biotinylated macromolecules via avidin-biotin interaction is described. This method better preserved functionality than standard covalent coupling techniques using carbodiimide cross-linking chemistry. The controlled release of avidin from the silk surface was demonstrated by altering the adsorption parameters. Application of this technique to culturing human foreskin fibroblasts (hFFs) and human mesenchymal stem cells (hMSCs) on arginine-glycine-aspartic-acid-modified (RGD-modified) silk showed increased cell growth over a seven-day period. This technique provides a facile method for the versatile functionalization of silk materials for biomedical applications including tissue engineering, drug delivery, and biological sensing.

Figure 1.. Schematic of conjugation and detection.

(A) The conjugation and detection method for modifying silk films with avidin, which then interacts with biotinylated molecules, and (B) the conjugation and detection method for modifying silk films with EDC/NHS carbodiimide coupling. pI indicates the isoelectric point of the molecule. Representative biotin structure used. Linker length of commercially provided product is 14 atoms. PDB file for the avidin structure used is 1VYO (DOI: 10.2210/pdb1VYO/pdb).

Figure 2.. Colorimetric characterization of avidin and B-HRP concentrations.

Avidin and B-HRP concentrations were varied to determine the optimal combination of concentrations. HRP was detected via the addition of a clear substrate (TMB) that is cleaved by HRP, forming a blue color. (A) Image of the array study where the triangles indicated the concentration gradients for each compound. A darker blue color indicates higher HRP activity correlating to increased avidin adsorption. (B) Quantification of the visible light absorbance indicating optimal avidin concentration at 1,000 μg/mL and optimal B-HRP concentration at 0.5 – 5 μg/mL.

Figure 3.. Kinetic curves of adsorbed or covalently coupled HRP moiety functionalized silk films.

Biotin-HRP or HRP (5 μg/mL) were adsorbed onto silk films modified with avidin or carboxylate activation. HRP activity was monitored via TMB cleavage forming a blue chromophore detected at 650 nm (15 s between each measurement). Data were normalized to first recorded value for each well. A faster rate of change (higher slope) indicates greater HRP activity. (A) All kinetic curves are displayed with the same y-axis for direct comparison. To more readily make comparisons (B) AB-H and E/NB-H are compared, (C) AH and E/NH are compared, and (D) B-H and H are compared. AB-H modified silk films had the highest HRP activity. Data are presented as mean ± SEM of three independent experiments with 3-6 samples each. Key: AB-H: Avidin with Biotin-HRP; AH: Avidin with HRP; E/NB-H: EDC/NHS with Biotin-HRP; E/NH: EDC/NHS with HRP; B-H: Biotin-HRP background control; H: HRP background control well.

Figure 4.. A-HRP release from silk films over time.

Rate of release of A-HRP from silk film for different time intervals sampled. Rates were determined from time points in the linear region of A-HRP activity curve using TMB as the substrate. Detectable quantities of desorbed A-HRP were observed for both group studies. When A-HRP was adsorbed overnight (black bars) versus for 1-hour (white bars), increased levels of HRP were detected after 19.75 h of release. Data were normalized to first recorded value for each well and are representative of three independent experiments with 8 samples per time point (**p < 0.01, ***p < 0.001 when comparing overnight to 1-hour samples).

Figure 5.. Metabolic activity of hFF on modified silk films.

Normalized fluorescence of resazurin after incubation with hFF cells following (A) 1 d, (B) 3 d, (C) or 7 d of culture. Data shown as average with SEM (N = 5 with 4 samples for experimental groups and 2 samples for protein binding, *p < 0.05; **p < 0.01 as compared to silk only controls). All values normalized to the average fluorescence for day 1 silk only wells. H indicates 10⁻⁷ M. L indicates 10⁻⁸ M. + indicates inclusion, − indicates exclusion. TCP is tissue culture-treated plastic.

Figure 6.. Metabolic activity of hMSCs on modified silk films.

Normalized fluorescence of resazurin incubated with hMSCs after 7 d of culture. Data shown as average with standard deviation from a single experiment with four samples. All values are normalized to the average fluorescence for hMSCs cultured for 1 day on silk only wells (*p < 0.05; **p < 0.01 as compared to silk only controls). H indicates 10⁻⁷ M. L indicates 10⁻⁸ M. + indicates inclusion, − indicates exclusion. TCP is tissue culture-treated plastic.