Functionalising silk hydrogels with hetero- and homotypic nanoparticles

Abstract

Despite many reports detailing silk hydrogels, the development of composite silk hydrogels with homotypic and heterotypic silk nanoparticles and their impact on material mechanics and biology have remained largely unexplored. We hypothesise that the inclusion of nanoparticles into silk-based hydrogels enables the formation of homotropic and heterotropic material assemblies. The aim was to explore how well these systems allow tuning of mechanics and cell adhesion to ultimately control the cell-material interface. We utilised nonporous silica nanoparticles as a standard reference and compared them to nanoparticles derived from Bombyx mori silk and Antheraea mylitta (tasar) silk (approximately 100-150 nm in size). Initially, physically cross-linked B. mori silk hydrogels were prepared containing silica, B. mori silk nanoparticles, or tasar silk nanoparticles at concentrations of either 0.05% or 0.5% (w/v). The initial modulus (stiffness) of these nanoparticle-functionalised silk hydrogels was similar. Stress relaxation was substantially faster for nanoparticle-modified silk hydrogels than for unmodified control hydrogels. Increasing the concentrations of B. mori silk and silica nanoparticles slowed stress relaxation, while the opposite trend was observed for hydrogels modified with tasar nanoparticles. Cell attachment was similar for all hydrogels, but proliferation during the initial 24 h was significantly improved with the nanoparticle-modified hydrogels. Overall, this study demonstrates the manufacture and utilisation of homotropic and heterotropic silk hydrogels.

Fig. 1. Flow diagram of nanoparticle manufacture. Silica, B. mori and tasar silk nanoparticle synthesis.

Fig. 2. Nanoparticle characterisation of silica, B. mori silk, and A. mylitta (tasar) silk nanoparticles. (A) Particle diameter (measured by dynamic light scattering [DLS]), polydispersity (PDI) and zeta potential values. Data are presented as mean ± SD, n = 6 independent measurements, (B) surface area and pore volume measurements using nitrogen adsorption. n = 1 from 6 pooled batches, and (C) morphology of nanoparticles by scanning electron microscopy (scale bar, 4 μm; zoom, 2 μm). One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test, ns: not significant.

Fig. 3. Fourier-transform infrared (FTIR) spectra and peak assignments. (A) FTIR spectra of B. mori and tasar silk nanoparticles, silk hydrogels, air-dried silk film (negative control), and 70% ethanol-treated silk film (positive control). (B) Analysed secondary structure.

Fig. 4. Impact of the nanoparticle concentration and type on silk hydrogel rheological properties. (A) Rheological behaviour of 3% w/v silk hydrogels doped with nanoparticles, (B) stress relaxation time and normalised stress relaxation time. Control refers to an undoped silk hydrogel. Data are presented as mean ± SD, n = 3 independent experiments. Closed symbols G′ and open symbols G′′.

Fig. 5. Silk hydrogels are used for cell culture studies. Monitoring DU145 cell attachment using PicoGreen kit assay at 2, 4 and 24 h. Control: silk hydrogel. TCP: tissue culture treated polystyrene. Data are presented mean ± SD, n = 3 independent biological experiments. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test.