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- ItemThe innate immune response of self-assembling silk fibroin hydrogels(Cambridge : Royal Soc. of Chemistry, 2021) Gorenkova, Natalia; Maitz, Manfred F.; Böhme, Georg; Alhadrami, Hani A.; Jiffri, Essam H.; Totten, John D.; Werner, Carsten; Carswell, Hilary V. O.; Seib, F. PhilippSilk has a long track record of use in humans, and recent advances in silk fibroin processing have opened up new material formats. However, these new formats and their applications have subsequently created a need to ascertain their biocompatibility. Therefore, the present aim was to quantify the haemocompatibility and inflammatory response of silk fibroin hydrogels. This work demonstrated that self-assembled silk fibroin hydrogels, as one of the most clinically relevant new formats, induced very low blood coagulation and platelet activation but elevated the inflammatory response of human whole blood in vitro. In vivo bioluminescence imaging of neutrophils and macrophages showed an acute, but mild, local inflammatory response which was lower than or similar to that induced by polyethylene glycol, a benchmark material. The time-dependent local immune response in vivo was corroborated by histology, immunofluorescence and murine whole blood analyses. Overall, this study confirms that silk fibroin hydrogels induce a similar immune response to that of PEG hydrogels, while also demonstrating the power of non-invasive bioluminescence imaging for monitoring tissue responses. This journal is
- 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.
- ItemMicrofluidic-assisted silk nanoparticle tuning(Cambridge : Royal Society of Chemistry, 2019) Wongpinyochit, Thidarat; Totten, John D.; Johnston, Blair F.; Seib, F. PhilippSilk is now making inroads into advanced pharmaceutical and biomedical applications. Both bottom-up and top-down approaches can be applied to silk and the resulting aqueous silk solution can be processed into a range of material formats, including nanoparticles. Here, we demonstrate the potential of microfluidics for the continuous production of silk nanoparticles with tuned particle characteristics. Our microfluidic-based design ensured efficient mixing of different solvent phases at the nanoliter scale, in addition to controlling the solvent ratio and flow rates. The total flow rate and aqueous : solvent ratios were important parameters affecting yield (1 mL min−1 > 12 mL min−1). The ratios also affected size and stability; a solvent : aqueous total flow ratio of 5 : 1 efficiently generated spherical nanoparticles 110 and 215 nm in size that were stable in water and had a high beta-sheet content. These 110 and 215 nm silk nanoparticles were not cytotoxic (IC50 > 100 μg mL−1) but showed size-dependent cellular trafficking. Overall, microfluidic-assisted silk nanoparticle manufacture is a promising platform that allows control of the silk nanoparticle properties by manipulation of the processing variables.
- 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.
- ItemMetabolic Reprogramming of Macrophages Exposed to Silk, Poly(lactic-co-glycolic acid), and Silica Nanoparticles(Weinheim : Wiley-VCH, 2017) Saborano, Raquel; Wongpinyochit, Thidarat; Totten, John D.; Johnston, Blair F.; Seib, F. Philipp; Duarte, Iola F.Monitoring macrophage metabolism in response to nanoparticle exposure provides new insights into biological outcomes, such as inflammation or toxicity, and supports the design of tailored nanomedicines. This paper describes the metabolic signature of macrophages exposed to nanoparticles ranging in diameter from 100 to 125 nm and made from silk, poly(lactic-co-glycolic acid) or silica. Nanoparticles of this size and type are currently at various stages of preclinical and clinical development for drug delivery applications. 1H NMR analysis of cell extracts and culture media is used to quantify the changes in the intracellular and extracellular metabolomes of macrophages in response to nanoparticle exposure. Increased glycolytic activity, an altered tricarboxylic acid cycle, and reduced ATP generation are consistent with a proinflammatory phenotype. Furthermore, amino acids possibly arising from autophagy, the creatine kinase/phosphocreatine system, and a few osmolytes and antioxidants emerge as important players in the metabolic reprogramming of macrophages exposed to nanoparticles. This metabolic signature is a common response to all nanoparticles tested; however, the direction and magnitude of some variations are clearly nanoparticle specific, indicating material-induced biological specificity. Overall, metabolic reprogramming of macrophages can be achieved with nanoparticle treatments, modulated through the choice of the material, and monitored using 1H NMR metabolomics.