(395c) Fabrication and Characterization of Novel Plodia Interpunctella Silk Fibroin-Based Regenerated Solutions and Films for Biomedical Applications

Biomaterial design has been a focus in medicine for tissue engineering and drug delivery applications, but recent advances have also favored applications of biomaterials in the food and agriculture industries. The need for items such as edible food coatings, food spoilage detection sensors, and materials to deliver payloads with controlled release has brought research into the environmental sphere. Silk-based biomaterials, primarily derived from the silk fibroin protein of the Bombyx mori (B. mori) mulberry silkworm, have been studied due to their advantageous mechanical properties, biocompatibility, and commercial availability.¹ The use of alternative silk sources, such as the Antheraea yamamai silk moth and Nephila clavipes spider, in biomaterial fabrication is limited by reliable silk sources with controlled growth environments and methods to produce aqueous, regenerated silk fibroin solution as a precursor to form materials such as films, particles, and hydrogels.^{2, 3} For the development of alternative silk biopolymer-based materials, we investigate a novel source of silk from the Plodia interpunctella (P. interpunctella) silkworm, a small agricultural pest that infests and damages food products via silk production that traps waste within stored goods. P. interpunctella can be cultivated in a lab setting with control over environmental parameters that impact silk production and properties (i.e., temperature, humidity, diet) to produce silk in scalable quantities (Figure 1A). Additionally, P. interpunctella silk fibers can be solubilized into regenerated silk solution using methods inspired by B. mori and Vespa simillima hornet silk protocols (Figure 1B).^{4, 5} Early experiments in cell viability and cytotoxicity to cancer cells have shown potential for P. interpunctella in regenerative medicine, however, P. interpunctella silk has not been largely characterized on the fiber or material level.⁶

In this work, we establish methods to solubilize P. interpunctella silk fibers, describe the formation and characterization of silk fibroin-based thin films, and assess the potential of P. interpunctella materials as an alternative to existing B. mori biomaterial formulations. In future applications, we aim to leverage the differences in primary and secondary structure between P. interpunctella and B. mori silk fibroin proteins to form biomaterials with a range of mechanical properties (e.g., tensile strength, toughness, elongation) and chemistries to tailor release of payloads to specific conditions.

Methods: Regenerated silk solution from B. mori silk is prepared by previously described methods.⁴ P. interpunctella silk fibers are dissolved in lithium bromide salt solution, filtered to remove insect waste and food contaminants, and dialyzed in water for 48 hours. Dissolution time of silk fibers was changed by manipulation of salt concentration (7, 7.5, 8, 8.5, 9, 9.3 M), temperature of dissolution (25 to 60 ËšC), and silk to salt solution ratios (g:ml - 1:20, 1:40, 1:60 1:80, 1:100). The molecular weight distribution of the silk solutions was measured by SDS-PAGE. To test for shifts in viscosity as a function of silk concentration, we measured solution viscosity using a cone and plate geometry on a rheometer at various rates of strains and constant temperatures (Figure 1C).

Films are formed by casting regenerated silk solution upon PDMS substrates (rinsed in 70% ethanol and DI water) 10 mm in diameter and drying samples overnight. Film cross sections were imaged by SEM to assess film thickness and morphology as a function of silk concentration (Figure 1D). To investigate film microstructure, films were water annealed or heat treated to induce formation of β-sheet structures and analyzed using micro-attenuated total reflection (Micro ATR) FTIR spectroscopy (4000-500 cm^-1 wavenumber range). Deconvolution of the amide I region into relative amounts of side chain, β-sheet, a-helix, random coil, and turn structures was performed as previously described.⁷ To assess thermal transitions within silk fibers and thin films, thermal gravimetric analysis was used to determine mass loss and decomposition temperatures over a heating range from room temperature to 400 ËšC.

Outcomes: P. interpunctella silk fibroin solutions and thin films can be formed using comparable methods to B. mori protocols. Higher temperatures of dissolution (60ËšC) led to decreased solubility of P. interpunctella silk in salt solution. It is hypothesized that this change from B. mori protocols stems from different thermal stability of the silk fibroin proteins. P. interpunctella films demonstrated lower β-sheet content than B. mori materials, as expected by known differences in silk fibroin protein sequence⁸ and fiber structure. Future work will investigate the fabrication of other biomaterials, specifically micro- and nanoparticles, using regenerated P. interpunctella silk fibroin solution and their applications within environmental and biomedical fields.

References: (1) Stoppel, W. 2.12 Silk Biomaterials. 2017. (2) Jo, Y.-Y. International Journal of Industrial Entomology 2017, 34 (1), 6-10. (3) Widhe, M. Biopolymers 2012, 97 (6), 468-478. (4) Rockwood, D. N. Nature protocols 2011, 6 (10), 1612-1631. (5) Kameda, T. Zeitschrift für Naturforschung C 2005, 60 (11-12), 906-914. (6) Milutinović, M. Animal biotechnology 2020, 31 (3), 195-202. (7) Hu, X. Macromolecules 2006, 39 (18), 6161-6170. (8) Childers, A. K. Insects 2021, 12 (7), 626. DOI: 10.3390/insects12070626 PubMed.