Optimizing protein delivery rate from silk fibroin hydrogel using silk fibroin-mimetic peptides conjugation

Abstract

Controlled release of proteins, such as growth factors, from biocompatible silk fibroin (SF) hydrogel is valuable for its use in tissue engineering, drug delivery, and other biological systems. To achieve this, we introduced silk fibroin-mimetic peptides (SFMPs) with the repeating unit (GAGAGS)_n. Using green fluorescent protein (GFP) as a model protein, our results showed that SFMPs did not affect the GFP function when conjugated to it. The SFMP-GFP conjugates incorporated into SF hydrogel did not change the gelation time and allowed for controlled release of the GFP. By varying the length of SFMPs, we were able to modulate the release rate, with longer SFMPs resulting in a slower release, both in water at room temperature and PBS at 37 °C. Furthermore, the SF hydrogel with the SFMPs showed greater strength and stiffness. The increased β-sheet fraction of the SF hydrogel, as revealed by FTIR analysis, explained the gel properties and protein release behavior. Our results suggest that the SFMPs effectively control protein release from SF hydrogel, with the potential to enhance its mechanical stability. The ability to modulate release rates by varying the SFMP length will benefit personalized and controlled protein delivery in various systems.

Figure 1

Schematics of the SFMPs. (A) The production of SFMP-conjugated protein used in this study ((GAGAGS)_n-GFP). (B) The design concept illustrating the possible interaction between the SFMPs and the crystalline domains that should hinder the initial burst release by diffusion and result in sustained release of the GFP.

Figure 2

Purity of (GAGAGS)_n-GFP proteins. SDS-PAGE analysis of the purified (GAGAGS)_n-GFP where n equals the number of SF repeats. 0R = unconjugated GFP, nR = GFP conjugated to n repeat of GAGAGS. Each lane contained 12 µg of protein.

Figure 3

Hydrogel morphology and gelation. (A) SF hydrogel (SF) and SF hydrogel containing (GAGAGS)₆-GFP (6R-GFP) under white light (left), or UV light (right). The scale was in centimeters. (B) Gelation time of different hydrogels. The experiment was done in triplicate. The error bars are standard deviations.

Figure 4

In vitro release profiles of the recombinant GFP from the SF hydrogels in different conditions. The release was studied in water at room temperature (left) and in PBS at 37 °C (right). (A,B) The release profile of the recombinant GFP in 7 days. (C,D) The release profiles during the first 2 h, magnified from (A) and (B), respectively. The experiment was done in triplicate. The error bars are standard deviations.

Figure 5

Release kinetics of the recombinant GFP released from the SF hydrogels in different conditions. The release was studied in water at room temperature (left) and in PBS at 37 °C (right). The release profiles were fitted to different kinetic models. (A,B) First-order model. (C,D) Pseudo-second-order model. (E,F) Korsmeyer-Peppas model. (G,H) Higuchi model.

Figure 6

Mechanical properties of hydrogels. (A) Representative stress–strain profiles of the hydrogels. (B) Young’s modulus. (C) Yield strength. (D) Yield strain. The experiment was done in triplicate. Each bar represents mean ± SD. *p < 0.05 versus unconjugated GFP (0R).

Figure 7

Rheological analysis of hydrogels. (A) Viscoelastic property. The storage (G′) and loss (G″) moduli. (B) Thixotropic analysis of the hydrogels exposed to an alternate low–high shear-rate regime. The experiment was done in triplicate.

Figure 8

Protein secondary structures and crystallinity study. (A) Representative FTIR spectra of the hydrogels. The dotted line labelled random coil is at x = 1653 cm⁻¹, and the one labelled β-sheet is at x = 1622 cm⁻¹. (B) Fractions of the secondary structures in the hydrogels resulted from deconvolution and curve fitting of the FTIR spectra. The FTIR experiment was repeated twice. (C) Representative XRD spectra of the hydrogels. The 2θ of 20.7 and 24.6 (dotted lines) are assigned to the crystalline β-sheets. (D) The crystallinity indices calculated from the ratio between the area under the curve of the β-sheet crystalline region to the total diffraction area. The XRD experiment was repeated twice.

Figure 9

Biocompatibility of hydrogel. Cell viability after the hydrogels were exposed to unloaded SF hydrogel (SF) or SF hydrogel containing unconjugated GFP (0R), (GAGAGS)₃-GFP (3R), or (GAGAGS)₆-GFP (6R). Tissue culture plate was used as a control (TCP). The experiment was done in triplicate. Each bar represents mean ± SD.