(483e) Exploring the Structure-Function Relationship of Plodia Interpunctella Silk Fibers

Statement of Purpose: Silk fibroin (SF), obtained from Bombyx mori (B. mori) silkworm cocoons, is a natural polymer used in the formation of biomaterials due to its biocompatibility, controllable degradation rate, and tunable physical properties.¹ In SF biomaterials, tunable parameters, mainly crystalline content and organization, affect the mechanical and physical properties of the resulting structures.² Knowledge about the mechanical and thermal properties of the raw silk material gives insight into its structural organization as well as identifying potential applications for biomaterials. While B. mori cocoons are the main SF source due to their use in the textile industry, other silk sources are being identified and characterized to utilize differences in protein expression among species to target specific applications. We have identified Plodia interpunctella (P. interpunctella), an agricultural pest, as an alternative silk source³ as it has different hypothesized crystallinity organization (based on gene sequences⁴), yet characterization of the fibers is limited. In P. interpunctella, we hypothesize that the decrease in β-sheet content, as compared to other silk fibroins like B. mori, and the increased spacing between its repeat units,^{4, 5} will produce a greater viscoelastic response and decreased degradation temperature compared to B. mori. Previous work characterizing P. interpunctella has been limited in methodology and sample size^{3, 5} and has focused on maximizing silk fiber production³ rather than analysis of the structure-function relationship of P. interpunctella silk fibroin and silk fibers for future uses. Here we report fully characterized structural, thermal, and mechanical properties of P. interpunctella raw and processed silk for different scales of isotropic sheets and aligned fiber structures to help inform future applications.

Methods: We generated improved reproducibility and sample size for different structural levels (Figure 1A) of analysis and isolation of silk fibroin from P. interpunctella. Degumming methods of boiling the raw silk in water for 15 minutes to isolate silk fibroin were used to compare shifts in physical properties as compared to the unprocessed (non-degummed) silk. Scanning electron microscopy (SEM) imaging was used to assess sheet thickness, fiber dimensions, and confirm the extent of degumming (Figure 1B-C). Secondary structure and total crystallinity content was assessed with Fourier transform infrared (FTIR) spectroscopy (n=6) by deconvolution of the amide I regions. Thermal properties were assessed via thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) to evaluate degradation and transition temperatures respectively (n=3). We performed bulk tensile mechanical assessments (n=6) on non-degummed (raw) and degummed (processed) silk fibroin sheets collected directly from the P. interpunctella insect rearing containers as well as aligned fiber bundles produced by the insect to determine the impact of anisotropy on P. interpunctella silk. To improve reproducibility, we secured the isotropic sheets and aligned fiber bundles to paper frames with a gauge length of 10 mm. The bulk mechanical properties were assessed using static testing on a dynamic mechanical analyzer (DMA) instrument at two different strain rates (1 mm/min and 10 mm/min). Young’s modulus, ultimate tensile strength, and strain at break were analyzed from the resulting stress-strain curves.

Results: In silk literature, it has been hypothesized that the thermal and mechanical response of the material is directly related to its protein sequence and resulting secondary structure, with emphasis given to the frequency of the repeat unit structure correlating to mechanical strength.^{5, 6} Analysis of P. interpunctella SF gene sequences suggests that the protein likely has shorter repeating motifs with irregular spacing,⁴ compared to B. mori. We were able to show, through numerous material characterization techniques, that these hypotheses hold when comparing P. interpunctella silk to other known silk types. We confirmed through FTIR that P. interpunctella has around 10% less β-sheet content than the traditional B. mori SF (Figure 1D). P. interpunctella fiber diameters (2.1 ± 0.5 μm) were found to be much smaller than other silk producing insects due to the small insect size. As expected, thermal properties, such as melting temperature (284.4 ± 1.9 °C) were found to be lower in P. interpunctella (Figure 1E) due to the lower β-sheet content. When compared to other silk fibers, P. interpunctella has much lower values for extensional modulus and ultimate tensile strength due to its protein secondary structure. Young’s modulus (Figure 1F) of non-degummed P. interpunctella fiber bundles (536 ± 127 MPa, 10 mm/mm) are around an order of magnitude lower in value than that of B. mori, the gold standard in the silk field. While we found that degumming increases the magnitude of Young’s modulus values of P. interpunctella (3052 ± 965 MPa, 10 mm/mm), they are still around 3-times smaller than the other silk types. We contribute these differences to the secondary structure of P. interpunctella silk. The lower β-sheet content and less repetitive structure were expected to produce more elastic and responsive materials rather than higher strength as seen in other silks and even B. mori to some extent. The use of P. interpunctella silk fibroin will help fill the gap in areas where more elastic natural materials are needed for biomedical applications, such as soft aligned musculoskeletal tissues. Future work will aim to process P. interpunctella in different material formats such as 3D sponges, nanoparticles, and electrospun mats. The structure-function relationship described here will inform the fabrication parameters necessary for biomaterial translation of P. interpunctella silk.

References:

1. D. N. Rockwood, R. C. Preda, T. Yücel, X. Wang, M. L. Lovett and D. L. Kaplan, Nature Protocols, 2011, 6, 1612-1631.
2. E. L. Aikman, A. P. Rao, Y. Jia, E. E. Fussell, K. E. Trumbull, J. Sampath and W. L. Stoppel, Journal of Biomedical Materials Research Part A, 2024, DOI: 10.1002/jbm.a.37703.
3. B. D. Shirk, I. Torres Pereira Meriade Duarte, J. B. McTyer, L. E. Eccles, A. H. Lateef, P. D. Shirk and W. L. Stoppel, ACS Biomater Sci Eng, 2024, DOI: 10.1021/acsbiomaterials.3c01372.
4. A. Y. Kawahara, C. G. Storer, A. Markee, J. Heckenhauer, A. Powell, D. Plotkin, S. Hotaling, T. P. Cleland, R. B. Dikow, T. Dikow, R. B. Kuranishi, R. Messcher, S. U. Pauls, R. J. Stewart, K. Tojo and P. B. Frandsen, GigaByte, 2022, 2022, gigabyte64.
5. R. Fedič, M. Žurovec and F. Sehnal, Journal of Biological Chemistry, 2003, 278, 35255-35264.
6. J. F. Jameson, M. O. Pacheco, J. E. Butler and W. L. Stoppel, Front Bioeng Biotech, 2021, 9, 664306.