Recombinant spider silk protein hydrogels

Earlam, Rowan (2021) Recombinant spider silk protein hydrogels. PhD thesis, University of Nottingham.

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Abstract

Protein hydrogels are hydrophilic biomaterials that swell and have material properties that resemble biological soft tissue. Hydrogels are appropriate for use in local drug delivery systems due to their extensive porous structure allowing diffusion. Hydrogels prepared from ex vivo animal derived proteins bring risk of pathogens and ethical concerns. These issues can be addressed by using recombinantly produced spider silk. Spider silk protein (spidroin) is non-immunogenic, biodegradable and has impressive material properties such as strength and elasticity making it a promising material for hydrogel biomedical applications. In this study the recently reported highly expressing, recombinant mini-spidroin NT2RepCT was processed for the first time into natural protein hydrogels and characterised as a local and controlled drug delivery system.

NT2RepCT self-assembled into physical hydrogels at 2.4 % (w/v) upon incubation at 37 °C in double distilled milli Q (MQ) water Tris, HEPES and CAPS buffers at pH 5.5, 7 and 10. The hydrogel’s optical properties, swelling behaviour, pore morphology, nano-fibril structures, stiffness and rheological properties were tunable depending on gelation conditions. It was found NT2RepCT hydrogels formed at pH 10 had increased swelling, high light transmittance and increased stiffness compared to those formed at pH 7 or in MQ water. Glutaraldehyde chemical crosslinking was utilised to increase NT2RepCT hydrogel stiffness.

The effect of hydrogel sample preparation ahead of scanning electron microscopy (SEM) on artefacts and secondary pores was investigated in cryo-SEM, high vacuum SEM and environmental SEM due to hydrogels high water content. It was found that the commonly used plunge freezing in liquid nitrogen technique created the most artefacts compared to metal mirror (slam) freezing and in situ lyophilisation exemplifying the need for careful hydrogel SEM sample preparation.

The NT2RepCT hydrogels formed at pH 10 and in MQ water were injectable and showed shear-thinning properties. These hydrogels were assessed as a controlled drug delivery system and showed pH dependent release of the model drug surrogate rhodamine B. Tyrosine conjugation of 4-(4-(2-azidoethoxy)phenyl)-1,2,4-triazolidine-3,5-dione (PTAD-azide) added a functional handle and the model drug surrogate fluorescein (FAM) was ‘clicked’ via copper catalyzed azide-alkyne cycloaddition. Improved and controlled release was seen in hydrogels containing conjugated FAM compared to hydrogels containing non-conjugated ‘free’ FAM.

The properties of the NT2RepCT hydrogels were similar to previously reported natural and recombinant silk hydrogels but without the need for high temperatures, denaturing purification conditions and extensive processing. This study illustrates the diversity and tunability of recombinant minispidroin NT2RepCT hydrogels and demonstrates their suitability as a highly transparent, injectable, pH sensitive and controlled hydrogel drug delivery system.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Thomas, Neil
Goodacre, Sara
Keywords: Protein hydrogels; Spider Silk; Miniature Spidroin; NT2RepCT; rheology; SEM; drug delivery; Biomaterials
Subjects: R Medicine > R Medicine (General) > R855 Medical technology. Biomedical engineering. Electronics
Faculties/Schools: UK Campuses > Faculty of Science > School of Chemistry
Item ID: 65608
Depositing User: Earlam, Rowan
Date Deposited: 04 Jul 2021 04:40
Last Modified: 04 Aug 2021 04:42
URI: http://eprints.nottingham.ac.uk/id/eprint/65608

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