Silk fibroin nanoparticles : in vitro performance of a putative anticancer nanomedicine

Despite the advantages of nanoparticle-based carriers for anticancer drug delivery, their translation into the clinic has been limited by factors including: (i) poor endocytic uptake and intracellular routing, (ii) macrophage clearance and (iii) a disregard of the tumour microenvironment governing nanoparticle uptake. As a result, there is a continued demand to explore the performance of polymer-based nanoparticles.;The principle hypothesis of this thesis is that silk fibroin nanoparticles can be used as anticancer nanomedicines. To validate this, the mechanisms governing drug release from silk fibroin nanoparticles are explored in Chapter 3. Next, the immunogenicity of silk fibroin nanoparticles towards macrophages is assessed (Chapter 4). Finally, Chapter 5 investigates the endocytosis of silk fibroin nanoparticles in response to the cell cycle and culture substrate mechanics.;This thesis provided the first experimental proof of lysosomotropic anticancer drug delivery from silk fibroin nanoparticles in single human breast cancer cells (Totten et al. 2017. J. Drug Target. 25, pp 865-872) (Chapter 3). Drug loaded silk fibroin nanoparticles were endocytosed by MCF-7 cells and a combination of the acidic lysosomal pH and enzymatic degradation facilitated drug release and subsequent nuclear translocation of the payload within 5 hours of dosing.;Next, nanoparticle-macrophage interactions were studied (Chapter 4). Silk fibroin nanoparticles exerted similar immunogenicity to silica and poly(lactic-co-glycolic acid) nanoparticles (Saborano, Wongpinyochit, Totten, Johnston, Seib and Duarte. 2017. Adv. Healthc. Mater. 6, 1601240). This indicated that silk fibroin nanoparticles can compete with leading healthcare materials in pre-clinical and clinical use.;Further assessment into immunomodulatory potential of silk fibroin nanoparticles revealed that they drive macrophage polarisation towards a pro-inflammatory M1-like state (Totten et al. 2019. ACS Appl. Mater. Interfaces. in press). This effect could be fine-tuned with surface modification (i.e. PEGylation). This observation is important because silk fibroin nanoparticles could act both as carriers for chemotherapeutics and as synergistic attenuators of tumour-associated macrophages in the tumour site.;Finally, advanced analysis of silk fibroin nanoparticle endocytosis was conducted (Chapter 5) by assessing intracellular trafficking in a time-dependent manner. Endocytosis of silk fibroin nanoparticles by breast cancer (MCF-7) cells was influenced by cell cycle progression, but not substrate mechanics. However, substrate mechanics were found to modulate the endocytic behaviour of healthy human (MCF-10A) breast epithelial cells. This relationship warrants further investigation with regard to the cellular response of nanomedicines.;Overall, this thesis accomplished in vitro analysis of silk fibroin nanoparticle drug delivery performance, macrophage interactions and endocytic uptake. These findings indicate that silk fibroin nanoparticles are emerging as an interesting biopolymer for anticancer applications. Work presented in this thesis provides a foundation to now move to pre-clinical in vivo studies.Despite the advantages of nanoparticle-based carriers for anticancer drug delivery, their translation into the clinic has been limited by factors including: (i) poor endocytic uptake and intracellular routing, (ii) macrophage clearance and (iii) a disregard of the tumour microenvironment governing nanoparticle uptake. As a result, there is a continued demand to explore the performance of polymer-based nanoparticles.;The principle hypothesis of this thesis is that silk fibroin nanoparticles can be used as anticancer nanomedicines. To validate this, the mechanisms governing drug release from silk fibroin nanoparticles are explored in Chapter 3. Next, the immunogenicity of silk fibroin nanoparticles towards macrophages is assessed (Chapter 4). Finally, Chapter 5 investigates the endocytosis of silk fibroin nanoparticles in response to the cell cycle and culture substrate mechanics.;This thesis provided the first experimental proof of lysosomotropic anticancer drug delivery from silk fibroin nanoparticles in single human breast cancer cells (Totten et al. 2017. J. Drug Target. 25, pp 865-872) (Chapter 3). Drug loaded silk fibroin nanoparticles were endocytosed by MCF-7 cells and a combination of the acidic lysosomal pH and enzymatic degradation facilitated drug release and subsequent nuclear translocation of the payload within 5 hours of dosing.;Next, nanoparticle-macrophage interactions were studied (Chapter 4). Silk fibroin nanoparticles exerted similar immunogenicity to silica and poly(lactic-co-glycolic acid) nanoparticles (Saborano, Wongpinyochit, Totten, Johnston, Seib and Duarte. 2017. Adv. Healthc. Mater. 6, 1601240). This indicated that silk fibroin nanoparticles can compete with leading healthcare materials in pre-clinical and clinical use.;Further assessment into immunomodulatory potential of silk fibroin nanoparticles revealed that they drive macrophage polarisation towards a pro-inflammatory M1-like state (Totten et al. 2019. ACS Appl. Mater. Interfaces. in press). This effect could be fine-tuned with surface modification (i.e. PEGylation). This observation is important because silk fibroin nanoparticles could act both as carriers for chemotherapeutics and as synergistic attenuators of tumour-associated macrophages in the tumour site.;Finally, advanced analysis of silk fibroin nanoparticle endocytosis was conducted (Chapter 5) by assessing intracellular trafficking in a time-dependent manner. Endocytosis of silk fibroin nanoparticles by breast cancer (MCF-7) cells was influenced by cell cycle progression, but not substrate mechanics. However, substrate mechanics were found to modulate the endocytic behaviour of healthy human (MCF-10A) breast epithelial cells. This relationship warrants further investigation with regard to the cellular response of nanomedicines.;Overall, this thesis accomplished in vitro analysis of silk fibroin nanoparticle drug delivery performance, macrophage interactions and endocytic uptake. These findings indicate that silk fibroin nanoparticles are emerging as an interesting biopolymer for anticancer applications. Work presented in this thesis provides a foundation to now move to pre-clinical in vivo studies

Silk nanoparticles : proof of lysosomotropic anticancer drug delivery at single cell resolution

Silk nanoparticles are expected to improve chemotherapeutic drug targeting to solid tumours by exploiting tumour pathophysiology, modifying the cellular pharmacokinetics of the payload and ultimately resulting in trafficking to lysosomes and triggering drug release. However, experimental proof for lysosomotropic drug delivery by silk nanoparticles in live cells is lacking and the importance of lysosomal pH and enzymes controlling drug release are currently unknown. Here, we demonstrate, in live single human breast cancer cells, the role of the lysosomal environment in determining silk nanoparticle-mediated drug release. MCF-7 human breast cancer cells endocytosed and trafficked drug-loaded native and PEGylated silk nanoparticles (approximately 100 nm in diameter) to lysosomes (n = 3), with subsequent drug release from the respective carriers and nuclear translocation within 5 h of dosing (n = 2). A combination of low pH and enzymatic degradation facilitated drug release from the silk nanoparticles (n = 3); perturbation of the acidic lysosomal pH and inhibition of serine, cysteine and threonine proteases resulted in a 42% ± 2.2% and 33% ± 3% reduction in nuclear-associated drug accumulation for native and PEGylated silk nanoparticles, respectively (n = 2). Overall, this study demonstrates the importance of lysosomal activity for anticancer drug release from silk nanoparticles, thereby providing direct evidence for lysosomotropic drug delivery in live cells

PEGylation-Dependent Metabolic Rewiring of Macrophages with Silk Fibroin Nanoparticles

Silk 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

Metabolic reprogramming of macrophages exposed to silk, poly(lactic-co-glycolic acid) and silica nanoparticles

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. We describe 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 pre-clinical and clinical development for drug delivery applications. We used 1H NMR analysis of cell extracts and culture media 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 were consistent with a pro-inflammatory phenotype. Furthermore, amino acids possibly arising from autophagy, the creatine kinase/phosphocreatine system and a few osmolytes and antioxidants emerged as important players in the metabolic reprogramming of macrophages exposed to nanoparticles. This metabolic signature was a common response to all nanoparticles tested; however, the direction and magnitude of some variations were 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

Manual versus microfluidic-assisted nanoparticle manufacture : impact of silk fibroin stock on nanoparticle characteristics

Silk 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 30 min) had no significant effect on particle attributes. Overall, the results showed that silk fibroin processing directly impacts nanoparticle characteristics

Silk Nanoparticle Manufacture in Semi-Batch Format

Silk 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

Silk nanoparticle manufacture in semi-batch format

Silk 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

Microfluidic-assisted silk nanoparticle tuning

Silk 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

The innate immune response of self-assembling silk fibroin hydrogels

Silk 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

Rapid retreat of Thwaites Glacier in the pre-satellite era

Understanding the recent history of Thwaites Glacier, and the processes controlling its ongoing retreat, is key to projecting Antarctic contributions to future sea-level rise. Of particular concern is how the glacier grounding zone might evolve over coming decades where it is stabilized by sea-floor bathymetric highs. Here we use geophysical data from an autonomous underwater vehicle deployed at the Thwaites Glacier ice front, to document the ocean-floor imprint of past retreat from a sea-bed promontory. We show patterns of back-stepping sedimentary ridges formed daily by a mechanism of tidal lifting and settling at the grounding line at a time when Thwaites Glacier was more advanced than it is today. Over a duration of 5.5 months, Thwaites grounding zone retreated at a rate of >2.1 km per year—twice the rate observed by satellite at the fastest retreating part of the grounding zone between 2011 and 2019. Our results suggest that sustained pulses of rapid retreat have occurred at Thwaites Glacier in the past two centuries. Similar rapid retreat pulses are likely to occur in the near future when the grounding zone migrates back off stabilizing high points on the sea floor