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.
Highlights
Nanoscale biomaterials is a rapidly expanding field of research that has seen great application success in areas such as cancer therapy [1,2], gene delivery [3,4], antimicrobial resistance [5,6], and tissue engineering [7,8]
We have previously demonstrated a multi-step chemical reaction process to modify the surface of a silica nanofiber for assisting cell attachment and enhancing neuronal cell growth and differentiation [30]
Successful production of the silica binding protein (SB)-RGD fusion proteins was achieved through isopropyl b-D-thiogalactopyranoside (IPTG) induction
Summary
Nanoscale biomaterials is a rapidly expanding field of research that has seen great application success in areas such as cancer therapy [1,2], gene delivery [3,4], antimicrobial resistance [5,6], and tissue engineering [7,8]. The neuronal tissue engineering field has especially focused on producing three-dimensional, bioactive, and biodegradable nanoscaffolds that mimic the extracellular matrix (ECM) [9] as a promising approach for nerve repair and nervous system regeneration [10,11]. Natural materials, such as collagen and laminin, could experience. In studying neuronal tissue engineering, an abundance of literature has highlighted the importance of providing surface contact guidance cues in the form of ECM proteins for developing neurons [16,17]. Within the ECM proteins, such as fibronectin, collagen, vitronectin, and laminin [18,19], an Arg-Gly-Asp (RGD) motif was found to improve cell adhesion to material surfaces [20] and is widely used for tissue engineering to promote cell growth [20,21,22].
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