Genetically Engineered Mucoadhesive Spider Silk

Abstract

Mucoadhesion is defined as the adhesion of a material to the mucus gel covering the mucous membranes. The mechanisms controlling mucoadhesion include nonspecific electrostatic interactions and specific interactions between the materials and the mucins, the heavily glycosylated proteins that form the mucus gel. Mucoadhesive materials can be used to develop mucosal wound dressings and noninvasive transmucosal drug delivery systems. Spider silk, which is strong, biocompatible, biodegradable, nontoxic, and lightweight would serve as an excellent base for the development of such materials. Here, we investigated two variants of the partial spider silk protein 4RepCT genetically engineered in order to functionalize them with mucoadhesive properties. The pLys-4RepCT variant was functionalized with six cationically charged lysines, aiming to provide nonspecific adhesion from electrostatic interactions with the anionically charged mucins, while the hGal3-4RepCT variant was genetically fused with the Human Galectin-3 Carbohydrate Recognition Domain which specifically binds the mucin glycans Galβ1-3GlcNAc and Galβ1-4GlcNAc. First, we demonstrated that coatings, fibers, meshes, and foams can be readily made from both silk variants. Measured by the adsorption of both bovine submaxillary mucin and pig gastric mucin, the newly produced silk materials showed enhanced mucin binding properties compared with materials of wild-type (4RepCT) silk. Moreover, we showed that pLys-4RepCT silk coatings bind mucins through electrostatic interactions, while hGal3-4RepCT silk coatings bind mucins through specific glycan-protein interactions. We envision that the two new mucoadhesive silk variants pLys-4RepCT and hGal3-4RepCT, alone or combined with other biofunctional silk proteins, constitute useful new building blocks for a range of silk protein-based materials for mucosal treatments.

Mucin CYS domain stiffens the mucus gel hindering bacteria and spermatozoa

“…Thereby, recombinant CYS domain delivery could help to determine the minimal amount of recombinant protein necessary to reinforce the colonic mucus with aim to limit diffusion of macromolecules and toxins in mucus as observed for the delivery of lipids 59 . Reinforcing mucus hydrogels is an attractive approach which can be achieved through the delivery of recombinant molecules 60 or by administering recombinant probiotics that would secrete molecules made of CYS domains. This strategy could help in reducing the bacterial burden in the context of mucus-associated diseases, such as ulcerative colitis 61 .…”

Design of Silk-Elastin-Like Protein Nanoparticle Systems with Mucoadhesive Properties

“…As previously described, SELPs containing arginine (R), lysine (K), glutamic acid (E), and cysteine (C) substitutes were selected for investigation as possible designs for mucoadhesive delivery systems, as these proteins contain charged groups and thiol groups resulting from the selected modification of the elastin "X" amino acid. Many studies have demonstrated the importance of electrostatic interactions in mucoadhesion [9,19,43,44]. Additionally, mucoadhesive nanoparticle drug delivery systems containing thiol groups have resulted in increased retention because of the propensity for disulfide bond formation with mucins containing thiol groups [44,45].…”

“…Despite the diverse and dynamic microenvironment, the mucosal surface provides numerous physical and chemical features that can be exploited to increase mucoadhesion of carrier particles in drug delivery systems [6][7][8]. These approaches include particles with tailored hydrogen-bonding, electrostatic interactions, hydrophilicity, thiolation, and specific binding to mucins, as well as precise control over size and shape of the substrate [9][10][11][12][13][14][15]. Numerous mucoadhesive designs have been developed to improve retention of particles in mucus membranes, such as lipids [16], lectins [17], chitosan [18], synthetic polymers [19], and other biopolymers [9,20] in the form of adhesive tablets [21], micro-and nanoparticles [22], films [23], nanogels [24], and sprays [25].…”

Artificial spider silk supports and guides neurite extension in vitro

“…Production of proteins in heterologous systems also comes with the benefit of allowing modifications of the produced proteins, such as covalently incorporated functional motifs. [25][26][27][28][29][30] Hence, recombinant spider silk fibers could both provide physical guidance for sprouting neurites as has been described for the native fiber, and be decorated with cell binding motifs providing signals that promote neurite extension. In addition to the relatively straightforward and feasible manufacturing process of recombinant spider silk fibers, such a scaffold would potentially outcompete those made from, for example, vitronectin only since it cannot be spun into fibers and likely be more flexible and bioactive compared to fibers manufactured from synthetic polymers.…”

“…From a clinical point of view, however, recombinant spider silk would be superior to the natural fiber since it is of non‐animal origin and contains only a single defined protein in contrast to the natural fiber that is composed of many different proteins. Production of proteins in heterologous systems also comes with the benefit of allowing modifications of the produced proteins, such as covalently incorporated functional motifs 25–30 . Hence, recombinant spider silk fibers could both provide physical guidance for sprouting neurites as has been described for the native fiber, and be decorated with cell binding motifs providing signals that promote neurite extension.…”