Manipulating the water–air interface to drive protein assembly for functional silk-like fibroin fibre production

communications materials Article https://doi.org/10.1038/s43246-024-00722-x Manipulating the water–air interface to drive protein assembly for functional silk-like fibroin fibre production Check for updates 1,2 1234567890():,; 1234567890():,; Rafael O. Moreno-Tortolero Robert Walker3, Louise Serpell 1 1 1 3 , Juliusz Michalski , Eleanor Wells , Flora Gibb , Nick Skaer , , Chris Holland 5 & Sean A. Davis 1 4 Silk’s remarkable properties arise from its hierarchical structure, formed through natural transformation from an aqueous solution to a solid fibre driven by pH and flow stress under low-energy conditions. In contrast, artificial silk fabrication typically relies on extrusion-based methods using coagulating baths and unnatural solvents, limiting true biomimetic replication. Here, we find that native-like silk fibroin forms viscoelastic films at the air-water interface. Utilizing this, we demonstrate a mild, all-aqueous method to seamlessly pull silk-like fibres with co-aligned nanofibrillar bundles. The fiber structure transitioned from hexagonally packed β-solenoid units at low pulling speeds to β-sheetrich structures at higher speeds. Fibers pulled near physiological speeds (26.3 mm s-¹) exhibited optimal mechanical properties, with an elastic modulus of 8 ± 1 GPa and toughness of 8 ± 5 MJ m-³, comparable to natural silk. This platform also enables embedding nanoparticles and biologics, offering broad applications in sensors, biocatalysis, and tissue engineering, expanding the potential of silkbased composite materials. Silk fibroin has captivated researchers for generations owing to its remarkable mechanical properties and unique self-assembly behavior1. As a result, this natural process has proven inspirational for the production of a whole host of silk-like fibres from synthetic components2,3. However, unlike synthetic materials, silk fibroin undergoes a, not fully understood, programmed transition from a liquid aqueous solution to a solid state with minimal energy input, making it a fascinating subject of study4,5. Recent investigations have provided further insight into the molecular self-assembly mechanism of Lepidoptera silk fibroin, revealing its nanofibrillar structure in the Silk-I configuration and the intricate interactions driving its solidification6. At the macroscopic level, natural silk fibres are not extruded but rather pulled into shape through a process akin to pultrusion7,8. This pultrusive mechanism, observed in silkworms and spiders, underpins the biomechanical sophistication of silk production in nature9. Reconstituted silk fibroin (RSF), although often used as a precursor in biomimetic silk materials research, has some inherent limitations due to its reduced molecular weight and the lack of the N-terminal domain (NTD) responsible for pH-controlled supramolecular assembly10. As a result, RSF requires non-native conditions, such as organic solvents11, coagulation baths12, or prepolymerised aggregates13, for fibre formation14,15. However, these methods impose environmental stress on the protein and deviate from the natural spinning process where only minimal energy inputs are required to fabricate the silk fibre7,13,16. Interestingly, both silk fibroin and spidroins exhibit surface-active properties, rapidly forming elastic films at the water–air interface17,18. Although the surface activity of native or native-like fibroin remains underexplored, recent insights into the protein’s structural dynamics suggest that the water–air interface plays a crucial role in directing assembly invitro19. By leveraging this behaviour, our aim was to fabricate silk-like fibres with enhanced control and efficiency. In this study, we propose a novel approach to silk fibre fabrication that harnesses the interfacial self-assembly of silk fibroin and its sensitivity to stresses. The method was developed from observations made while working with dilute native-like silk fibroin (NLSF) solution (see ST 1 and Figures S1, S2). Notably, we overcame some key processing issues related to using native and native-like silk fibroin precursor solutions to produce fibrous materials. Concentrated fibroin solutions are highly shear sensitive20,21, and this property severely inhibits any attempt to mix and disperse dopants or combine with other phases. We circumvent this problem by using dilute solutions of the protein which tends to then 1 School of Chemistry, University of Bristol, Cantock’s Close, Bristol, UK. 2Max Planck-Bristol Centre for Minimal Biology, School of Chemistry, University of Bristol, Bristol, UK. 3Orthox Ltd; Milton Park, 66 Innovation Drive, Milton, Abingdon, UK. 4Sussex Neuroscience, School of Life Sciences, University of Sussex; Falmer, Brighton, UK. 5School of Chemical, Materials and Biological Engineering, University of Sheffield, Mappin Street, Sheffield, UK. e-mail: ; Communications Materials | (2024)5:277 1 Article https://doi.org/10.1038/s43246-024-00722-x Overall, our study seeks to deepen our understanding of silk fibroin assembly and pave the way for the development of biomimetic materials with tailored properties and applications. By bridging the gap between fundamental research and practical applications, we aim to unlock the full potential of silk fibroin as a versatile biomaterial. concentrate at the water–air interface. Moreover, additional dispersants can be easily introduced into the aqueous subphase and subsequently, readily incorporated into the resulting silk-fibre. This very mild approach, that takes advantage of the natural assembly properties of the protein, offers considerable benefits when producing living composites The use of low protein concentrations, and the absence of coagulating baths and organic solvents altogether offers an environment amenable to introducing living cell components, that would otherwise suffer from osmotic shocks (when using concentrated protein stocks or salt baths) or be exposed to toxic organic solvents (like hexafluoro isopropanol, a common solvent for silk and silk-like protein)22. In brief, fibroin molecules adsorb at the water–air interface and by applying perpendicular extension we induce a strain field. Pulling the film in this way promotes the more ordered alignment and registration of solenoid units via lateral interactions and subsequent consolidation leads to fibre formation. Our proposed molecular level mechanism is illustrated in Fig. 1 (Video 1). We believe this method offers a promising alternative to conventional methods, allowing to produce silk-like fibres under more physiologically relevant conditions. Although similar observations have also been reported for recombinant spidroin systems23,24, these have not recognised the role of the interface in driving assembly, nor more importantly have shown evidence of scalability of the process. Interfacial assembly of the protein at the water–air interface To better understand our initial observations and validate our proposed process, the surface assembly propertie (...truncated)