Silk use in biomedical products was only clinically approved in 2013 by FDA despite being used as early as 2000 BC [1]. Due to its loading applications in sutures, surgical mesh, and garments, silk is also used in advanced applications such as tissue engineering, disease models, implantable devices, and drug release [2–5].
Biomedical applications typically use silk fibroin (SF) gotten from Bombyx mori, a domesticated silkworm [6]. Bombyx mori silk is comprised of a core protein (SF) that is coated with silk sericin. SF has mainly a primary and secondary structure containing a light chain (L-chain, Mw ≈26 kDa) and a heavy chain (H-chain, Mw≈391 kDa) respectively. The SF secondary structure consists of crystalline structures; Silk I (α-helix) and Silk II (β -sheet) thus main distributes β-sheet structures in the SF nanoparticles [3, 7, 8]. β-sheets structures absorb impact pressures distributing them all over the fibroin fishnet network providing a better overall mechanical strength [9]. Furthermore, β -sheets are mainly used in silk nanofibrils and microfibrils showing no significant cytotoxicity toward in vitro neuronal cells thus being biocompatible[7]. β-sheets and disulfide bonds improve the responsive capacity of SF-based materials to acidity, reactive oxygen species, and glutathione that facilitate the on-demand release of loaded drugs from nanoparticles making SF a promising nano-carrier [3]. Controlling the SF secondary structure (Silk I and II) controls any adjustments to properties like biocompatibility, biodegradability, mechanical robustness, and durability [5, 7]. SF can be controlled during processing to produce β- rich sheets that provide properties needed for clinically accepted silk.
The clinically approved silk must undergo two crucial steps; complete removal of silk sericin and regeneration of silk fibroin. Complete and reproducible sericin removal is an essential step in SF utilization because silk sericin causes immune responses and also inhibits the conversion of soluble silk I (α-helix) into silk II (β-sheet-rich silk) [3, 8]. Degumming removes sericin and is done either by enzymatic methods (digesting sericin) or commonly used chemical processing (alkaline treatment) [4]. The remaining degummed silk is SF fibre which is essential to produce regenerated silk fibroin [7].
Regenerating SF, as the other critical step, mainly involves SF fibre dissolution. It should be noted that SF is mainly H-chains comprising of β-sheet structures making it highly crystalline. SF is also comprised of a large number of hydrogen bonds and 75% amino acids making it nonpolar and hydrophobic. As such, SF fibres are only soluble in solvents that break the hydrogen bonds and hydrophobic interactions which in turn break the peptide chains. The commonly recommended dissolution procedure involves the use of a high-concentration chaotropic agent like 9.3M lithium bromide at 60°C for 4 hours [4, 10] and high ionic aqueous or organic salt-containing systems such as CaCl2-Ethanol-H2O (Ajisawa’s ternary solvent system) [7, 8, 10] After dissolution, different regenerated SF formats are obtained for storage and further use.
Regenerated SF formats are limited by storage conditions and usage timelines and they include; an SF-solvent mixture, purified SF aqueous solution, SF gel, and SF powder. SF-solvent mixture is gotten immediately after dissolution while its dialysis against distilled water removes electrolytes leaving a purified SF aqueous solution [5]. Storage of purified SF aqueous solution is limited regardless of the temperature. For instance, it can be stored for a week at room temperature or 1 month at 4oC after which deterioration and degradation begin [10]. Purified SF aqueous solution is used to make SF gel which is equally stored at room temperature for not more than 7 days [4, 11]. Furthermore, purified SF aqueous solution when centrifuged and lyophilised leaves 100% SF powder which is stored indefinitely at room temperature and thus suitable for long-term storage. This powder is purer than SF fibres and the other regenerated SF formats [4]. It can easily be transformed into any SF products like gels, films, microspheres, sponges, electrospun materials, etc. [4, 5, 11]. However, purified SF powder is unpopular possibly to the needed extra steps of centrifugation, and lyophilization needed during its production [4]. Although purified SF aqueous solution is commonly used, it poses challenges in LMICs as it requires frequent processing of new batches because of its short shelf life. Furthermore, LMICs also face limited access to silk dissolution solvent alternatives. For instance, in Uganda, 100ml lithium bromide cost $69 in 2015 and by 2019, COVID-19, increased its costs and inaccessibility.
This research, therefore, seeks to develop a low-cost method for producing a β-sheet-packed silk fibroin powder for medical application in LMICs. It follows the registered protocol by Rockwood et al. (2013) [4]. However, lithium bromide was replaced with cheaper Ajisawa solution [12–14], formic acid as a solvent with lithium chloride and calcium chloride salts [15] for SF dissolution which was further processed into SF powder.