(209b) Understanding the Morphology and Controlled Release Potential of 3D Silk Fibroin Particle Laden Sponges to Direct Macrophage-Material Interactions

Biomaterials formed from the silk fibroin of Bombyx mori have proven valuable across several material formats including nano- and microparticles², films³, and sponge-like scaffolds⁴. Tunable characteristics including mechanics⁵, degradation timelines⁶, and cargo release profiles⁷ along with strong cytocompatibility and nonthrombogenic⁸ behavior make silk fibroin a biopolymer well suited for applications in tissue engineering and regenerative medicine. Previous work has demonstrated that the formation of β-sheet rich crystalline domains plays a central role in dictating the material properties and function of silk materials.⁶ In this work, we explore how interfacing two separate silk material formats (microparticles and sponges), each with unique crystal formation processes, impacts final material morphology, degradation, controlled release, and cell-material interactions. Recent work has demonstrated the benefits of particle-based scaffolds and hydrogels over bulk materials for improvements in nutrient diffusion, cellular infiltration, and injectability⁹. Particles in these systems are commonly covalently crosslinked to hold together the macroscale structure. In this work, we explore the potential of incorporating silk polymer to bind the structure together through formation of physical crosslinks. By tuning the ratio of silk fibroin microparticles (SFMPs) to free aqueous silk fibroin (SF) and adjusting the method of water vapor annealing, we control the interparticle connectivity and interfacial mechanics. We demonstrate that increased free SF and increased temperature during annealing lead to increased total β-sheet crystallinity and increased sponge-like morphology (Figure 1AB). The differences in crystal structure led to tunable enzymatic degradation rates as observed through continued assessment of mechanical properties and morphological changes. As the spongy matrix is degraded, particles are then liberated (Figure 1C). This process shows promise for the local delivery of multiple cargos at different time scales. To further explore this potential, we model the transport of the system in COMSOL® to determine what dynamics of release can be achieved (co-delivery, sequential delivery, etc.) as a function of material formulation and initial particle size. We then show the effectiveness of the system by the loading of IL-4 into the SFMPs and IFN-γ into the surrounding sponge matrix and monitoring the impact of the release overtime on the phenotype of RAW 264.7 macrophages. In wound healing, the dynamic phenotype of macrophages is critical for normal tissue repair. It is advantageous to have early activation of M1 macrophages, but for proper healing the switch to an M2 phenotype is critical.¹⁰ By loading the sponge matrix with IFN-γ (an M1 stimulating cytokine) and the particles with IL-4 (an M2 stimulating cytokine), we can monitor the impact of the sequential delivery on the phenotype of the macrophages in vitro through immunohistochemistry and flow cytometry. This study helps progress toward the rational design of multi-phase silk materials with predictable delivery cascades. Future work seeks to incorporate varied cargos into this material and confirm efficacy in both in vitro and in vivo models.

References

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