A Single-Step Surface Modification of Electrospun Silica Nanofibers Using a Silica Binding Protein Fused with an RGD Motif for Enhanced PC12 Cell Growth and Differentiation

Abstract

In this study, a previously known high-affinity silica binding protein (SB) was genetically engineered to fuse with an integrin-binding peptide (RGD) to create a recombinant protein (SB-RGD). SB-RGD was successfully expressed in Escherichia coli and purified using silica beads through a simple and fast centrifugation method. A further functionality assay showed that SB-RGD bound to the silica surface with an extremely high affinity that required 2 M MgCl2 for elution. Through a single-step incubation, the purified SB-RGD proteins were noncovalently coated onto an electrospun silica nanofiber (SNF) substrate to fabricate the SNF-SB-RGD substrate. SNF-SB-RGD was characterized by a combination of scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and immunostaining fluorescence microscopy. As PC12 cells were seeded onto the SNF-SB-RGD surface, significantly higher cell viability and longer neurite extensions were observed when compared to those on the control surfaces. These results indicated that SB-RGD could serve as a noncovalent coating biologic to support and promote neuron growth and differentiation on silica-based substrates for neuronal tissue engineering. It also provides proof of concept for the possibility to genetically engineer protein-based signaling molecules to noncovalently modify silica-based substrates as bioinspired material.

Engineering Biomimetic Extracellular Matrix with Silica Nanofibers: From 1D Material to 3D Network

“…213−216 The other is to attach matrix proteins such as laminin to the surfaces of SNFs. 16,139 4.2.1. Incorporation of Silica Nanoparticles in Fibrous Structures.…”

“…Comparably, directly incorporating matrix proteins such as laminin into SNFs can be much easier as fewer steps are involved. 16,139 In this work, 16 a high-affinity silica binding protein (SB) was genetically engineered to fuse with an integrin-binding peptide (RGD) to create a recombinant protein (SB-RGD). Then, they developed a method to anchor the SB-RGD onto silica surfaces in one step.…”

“…Chen et al identified a silica-binding protein, the Si-tag, 236 for a one-step anchoring of RGD onto silica surfaces. 16 They created a new fusion protein SB-RGD, the Si-tag for mediating SNF surface binding site and the RGD motif for integrin-receptor-based cell adherence. Their method avoids potential damage from covalent modification and provides a prospect for other protein-based signaling molecules for SNF ECM.…”

“…14 Therefore, biomaterials synthesized ECM using nanotechnologies has been widely utilized due to its high reproducibility from given chemical components 15 and easy modification to enhance cell growth. 16 Generally, there are two categories of ECM materials. The first is natural ECMs that are derived from living animals or plants such as collagen (typically type I or type II) and gelatin.…”

Unchain My Heart: Integrins at the Basis of iPSC Cardiomyocyte Differentiation

“…Human mesenchymal stem cells exhibit enhanced osteogenesis in response to high-strength binding which results from the activation of a YAP-mediated pathway [199]. This result suggests that traditional covalent biological coatings could generate a bias in data interpretation, which implies the need for novel coating methods, such as noncovalent coatings [200]. Based on these findings, it is clear that the study of integrin recruitment can not omit the consideration of forces acting on the whole chain, from the substrate, e.g., substrate rigidity to the engaged cytoskeleton filaments.…”