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- ItemIn Vivo Evaluation of Engineered Self-Assembling Silk Fibroin Hydrogels after Intracerebral Injection in a Rat Stroke Model(Washington, DC : ACS Publications, 2019) Gorenkova, Natalia; Osama, Ibrahim; Seib, F. Philipp; Carswell, Hilary V.O.Targeting the brain cavity formed by an ischemic stroke is appealing for many regenerative treatment strategies but requires a robust delivery technology. We hypothesized that self-assembling silk fibroin hydrogels could serve as a reliable support matrix for regeneration in the stroke cavity. We therefore performed in vivo evaluation studies of self-assembling silk fibroin hydrogels after intracerebral injection in a rat stroke model. Adult male Sprague-Dawley rats (n = 24) underwent transient middle cerebral artery occlusion (MCAo) 2 weeks before random assignment to either no stereotaxic injection or a stereotaxic injection of either self-assembling silk fibroin hydrogels (4% w/v) or PBS into the lesion cavity. The impact on morbidity and mortality, space conformity, interaction with glial scar, interference with inflammatory response, and cell proliferation in the lesion cavity were examined for up to 7 weeks by a blinded investigator. Self-assembling hydrogels filled the stroke cavity with excellent space conformity and presented neither an overt microglial/macrophage response nor an adverse morbidity or mortality. The relationship between the number of proliferating cells and lesion volume was significantly changed by injection of self-assembling silk hydrogels. This in vivo stroke model confirmed that self-assembling silk fibroin hydrogels provide a favorable microenvironment as a future support matrix in the stroke cavity. Copyright © 2018 American Chemical Society.
- ItemSilk Nanoparticle Manufacture in Semi-Batch Format(Washington, DC : ACS Publications, 2020) Matthew, Saphia A.L.; Totten, John D.; Phuagkhaopong, Suttinee; Egan, Gemma; Witte, Kimia; Perrie, Yvonne; Seib, F. PhilippSilk nanoparticles have demonstrated utility across a range of biomedical applications, especially as drug delivery vehicles. Their fabrication by bottom-up methods such as nanoprecipitation, rather than top-down manufacture, can improve critical nanoparticle quality attributes. Here, we establish a simple semi-batch method using drop-by-drop nanoprecipitation at the lab scale that reduces special-cause variation and improves mixing efficiency. The stirring rate was an important parameter affecting nanoparticle size and yield (400 < 200 < 0 rpm), while the initial dropping height (5.5 vs 7.5 cm) directly affected nanoparticle yield. Varying the nanoparticle standing time in the mother liquor between 0 and 24 h did not significantly affect nanoparticle physicochemical properties, indicating that steric and charge stabilizations result in high-energy barriers for nanoparticle growth. Manufacture across all tested formulations achieved nanoparticles between 104 and 134 nm in size with high β-sheet content, spherical morphology, and stability in aqueous media for over 1 month at 4 °C. This semi-automated drop-by-drop, semi-batch silk desolvation offers an accessible, higher-throughput platform for standardization of parameters that are difficult to control using manual methodologies. © 2020 American Chemical Society.
- ItemManual Versus Microfluidic-Assisted Nanoparticle Manufacture: Impact of Silk Fibroin Stock on Nanoparticle Characteristics(Washington, DC : ACS Publications, 2020) Solomun, Jana I.; Totten, John D.; Wongpinyochit, Thidarat; Florence, Alastair J.; Seib, F. PhilippSilk has a long track record of clinical use in the human body, and new formulations, including silk nanoparticles, continue to reveal the promise of this natural biopolymer for healthcare applications. Native silk fibroin can be isolated directly from the silk gland, but generating sufficient material for routine studies is difficult. Consequently, silk fibroin, typically extracted from cocoons, serves as the source for nanoparticle formation. This silk requires extensive processing (e.g., degumming, dissolution, etc.) to yield a hypoallergenic aqueous silk stock, but the impact of processing on nanoparticle production and characteristics is largely unknown. Here, manual and microfluidic-assisted silk nanoparticle manufacturing from 60-and 90-min degummed silk yielded consistent particle sizes (100.9-114.1 nm) with low polydispersity. However, the zeta potential was significantly lower (P < 0.05) for microfluidic-manufactured nanoparticles (-28 to-29 mV) than for manually produced nanoparticles (-39 to-43 mV). Molecular weight analysis showed a nanoparticle composition similar to that of the silk fibroin starting stock. Reducing the molecular weight of silk fibroin reduced the particle size for degumming times ≤30 min, whereas increasing the molecular weight polydispersity improved the nanoparticle homogeneity. Prolonged degumming (>30 min) had no significant effect on particle attributes. Overall, the results showed that silk fibroin processing directly impacts nanoparticle characteristics. Copyright © 2020 American Chemical Society.
- ItemPEGylation-Dependent Metabolic Rewiring of Macrophages with Silk Fibroin Nanoparticles(Washington, DC : ACS Publications, 2019) Totten, John D.; Wongpinyochit, Thidarat; Carrola, Joana; Duarte, Iola F.; Seib, F. PhilippSilk fibroin nanoparticles are emerging as promising nanomedicines, but their full therapeutic potential is yet to be realized. These nanoparticles can be readily PEGylated to improve colloidal stability and to tune degradation and drug release profiles; however, the relationship between silk fibroin nanoparticle PEGylation and macrophage activation still requires elucidation. Here, we used in vitro assays and nuclear magnetic resonance based metabolomics to examine the inflammatory phenotype and metabolic profiles of macrophages following their exposure to unmodified or PEGylated silk fibroin nanoparticles. The macrophages internalized both types of nanoparticles, but they showed different phenotypic and metabolic responses to each nanoparticle type. Unmodified silk fibroin nanoparticles induced the upregulation of several processes, including production of proinflammatory mediators (e.g., cytokines), release of nitric oxide, and promotion of antioxidant activity. These responses were accompanied by changes in the macrophage metabolomic profiles that were consistent with a proinflammatory state and that indicated an increase in glycolysis and reprogramming of the tricarboxylic acid cycle and the creatine kinase/phosphocreatine pathway. By contrast, PEGylated silk fibroin nanoparticles induced milder changes to both inflammatory and metabolic profiles, suggesting that immunomodulation of macrophages with silk fibroin nanoparticles is PEGylation-dependent. Overall, PEGylation of silk fibroin nanoparticles reduced the inflammatory and metabolic responses initiated by macrophages, and this observation could be used to guide the therapeutic applications of these nanoparticles. © 2019 American Chemical Society.
- ItemSilk Hydrogel Substrate Stress Relaxation Primes Mesenchymal Stem Cell Behavior in 2D(Washington, DC : ACS Publications, 2021) Phuagkhaopong, Suttinee; Mendes, Luís; Müller, Katrin; Wobus, Manja; Bornhäuser, Martin; Carswell, Hilary V.O.; Duarte, Iola F.; Seib, F. PhilippTissue-mimetic silk hydrogels are being explored for diverse healthcare applications, including stem cell delivery. However, the impact of stress relaxation of silk hydrogels on human mesenchymal stem cell (MSC) biology is poorly defined. The aim of this study was to fabricate silk hydrogels with tuned mechanical properties that allowed the regulation of MSC biology in two dimensions. The silk content and stiffness of both elastic and viscoelastic silk hydrogels were kept constant to permit direct comparisons. Gene expression of IL-1β, IL-6, LIF, BMP-6, BMP-7, and protein tyrosine phosphatase receptor type C were substantially higher in MSCs cultured on elastic hydrogels than those on viscoelastic hydrogels, whereas this pattern was reversed for insulin, HNF-1A, and SOX-2. Protein expression was also mechanosensitive and the elastic cultures showed strong activation of IL-1β signaling in response to hydrogel mechanics. An elastic substrate also induced higher consumption of glucose and aspartate, coupled with a higher secretion of lactate, than was observed in MSCs grown on viscoelastic substrate. However, both silk hydrogels changed the magnitude of consumption of glucose, pyruvate, glutamine, and aspartate, and also metabolite secretion, resulting in an overall lower metabolic activity than that found in control cells. Together, these findings describe how stress relaxation impacts the overall biology of MSCs cultured on silk hydrogels. ©