Silk nanoparticles - an emerging anticancer nanomedicine

Silk is a sustainable and ecologically friendly biopolymer with a robust clinical track record in humans for load bearing applications, in part due to its excellent mechanical properties and biocompatibility. Our ability to take bottom-up and top-down approaches for the generation of silk (inspired) biopolymers has been critical in supporting the evolution of silk materials and formats, including silk nanoparticles for drug delivery. Silk nanoparticles are emerging as interesting contenders for drug delivery and are well placed to advance the nanomedicine field. This review covers the use of Bombyx mori and recombinant silks as an anticancer nanomedicine, highlighting the emerging trends and developments as well as critically assessing the current opportunities and challenges by providing a context specific assessment of this multidisciplinary field

Silk hydrogels for drug and cell delivery

Silk has fascinated humans since ancient times; silk fibres have been used in textiles for more than 5,000 years and for many centuries as a suturing material (Lubec, Holbaubek et al. 1993, Omenetto and Kaplan 2010). The remarkable strength and toughness of silk stems from its evolution as a structural engineering material in nature (Vollrath and Porter 2009, Buehler 2013). Silk is a sustainable and ecologically benign biopolymer that can be manufactured using green processes (Vollrath and Porter 2009). Over the past 25 years, we have seen a tremendous development of both bottom-up and top-down approaches for the generation of silk biopolymers

Emerging silk material trends : repurposing, phase separation and solution-based designs

Silk continues to amaze. This review unravels the most recent progress in silk science, spanning from fundamental insights to medical silks. Key advances in silk flow are examined, with specific reference to the role of metal ions in switching silk from a storage to a spinning state. Orthogonal thermoplastic silk molding is described, as is the transfer of silk flow principles for the triggering of flow-induced crystallization in other non-silk polymers. Other exciting new developments include silk-inspired liquid–liquid phase separation for non-canonical fiber formation and the creation of "silk organelles" in live cells. This review closes by examining the role of silk fabrics in fashioning facemasks in response to the SARS-CoV-2 pandemic

Degradation behavior of silk nanoparticles – enzyme responsiveness

Silk nanoparticles are viewed as promising vectors for intracellular drug delivery as they can be taken up into cells by endocytosis and trafficked to lysosomes, where lysosomal enzymes and the low pH trigger payload release. However, the subsequent degradation of the silk nanoparticles themselves still requires study. Here, we report the responsiveness of native and PEGylated silk nanoparticles to degradation following exposure to proteolytic enzymes (protease XIV and α-chymotrypsin) and papain, a cysteine protease. Both native and PEGylated silk nanoparticles showed similar degradation behavior over a 20 day exposure period (degradation rate: protease XIV > papain >> chymotrypsin). Within 1 day, the silk nanoparticles were rapidly degraded by protease XIV, resulting in a ~50% mass loss, an increase in particle size, and a reduction in the amorphous content of the silk secondary structure. By contrast, 10 days of papain treatment was necessary to observe any significant change in nanoparticle properties, and chymotrypsin treatment had no effect on silk nanoparticle characteristics over the 20-day study period. Silk nanoparticles were also exposed ex vivo to mammalian lysosomal enzyme preparations to mimic the complex lysosomal microenvironment. Preliminary results indicated a 45% reduction in the silk nanoparticle size over a 5-day exposure. Overall, the results demonstrate that silk nanoparticles undergo enzymatic degradation, but the extent and kinetics are enzyme specific

Degradation Behavior of Silk Nanoparticles - Enzyme Responsiveness

Silk nanoparticles are viewed as promising vectors for intracellular drug delivery as they can be taken up into cells by endocytosis and trafficked to lysosomes, where lysosomal enzymes and the low pH trigger payload release. However, the subsequent degradation of the silk nanoparticles themselves still requires study. Here, we report the responsiveness of native and PEGylated silk nanoparticles to degradation following exposure to proteolytic enzymes (protease XIV and α-chymotrypsin) and papain, a cysteine protease. Both native and PEGylated silk nanoparticles showed similar degradation behavior over a 20 day exposure period (degradation rate: protease XIV > papain ≫ α-chymotrypsin). Within 1 day, the silk nanoparticles were rapidly degraded by protease XIV, resulting in a ∼50% mass loss, an increase in particle size, and a reduction in the amorphous content of the silk secondary structure. By contrast, 10 days of papain treatment was necessary to observe any significant change in nanoparticle properties, and α-chymotrypsin treatment had no effect on silk nanoparticle characteristics over the 20-day study period. Silk nanoparticles were also exposed ex vivo to mammalian lysosomal enzyme preparations to mimic the complex lysosomal microenvironment. Preliminary results indicated a 45% reduction in the silk nanoparticle size over a 5-day exposure. Overall, the results demonstrate that silk nanoparticles undergo enzymatic degradation, but the extent and kinetics are enzyme-specific

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

PEGylated silk nanoparticles for anticancer drug delivery

Silk has a robust clinical track record and is emerging as a promising biopolymer for drug delivery, including its use as nanomedicine. However, silk-based nanomedicines still require further refinements for full exploitation of their potential; the application of "stealth" design principals is especially necessary to support their evolution. The aim of this study was to develop and examine the potential of PEGylated silk nanoparticles as an anticancer drug delivery system. We first generated B. mori derived silk nanoparticles by driving β-sheet assembly (size 104 ± 1.7 nm, zeta potential -56 ± 5.6 mV) using nanoprecipitation. We then surface grafted polyethylene glycol (PEG) to the fabricated silk nanoparticles and verified the aqueous stability and morphology of the resulting PEGylated silk nanoparticles. We assessed the drug loading and release behavior of these nanoparticles using clinically established and emerging anticancer drugs. Overall, PEGylated silk nanoparticles showed high encapsulation efficiency (>93%) and a pH-dependent release over 14 days. Finally, we demonstrated significant cytotoxicity of drug loaded silk nanoparticles applied as single and combination nanomedicines to human breast cancer cells. In conclusion, these results, taken together with prior silk nanoparticle data, support a viable future for silk-based nanomedicines

In Vivo Evaluation of Engineered Self-Assembling Silk Fibroin Hydrogels after Intracerebral Injection in a Rat Stroke Model

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

Impact of the hypoxia phenotype on the uptake and efflux of nanoparticles by human breast cancer cells

Breast cancer cells adapt to the hypoxic tumoral environment by undergoing changes in metabolism, cell signalling, endo-lysosomal receptor uptake and recycling. The resulting hypoxic cell phenotype has the potential to undermine the therapeutic efficacy of nanomedicines designed for endocytic uptake and specific intracellular trafficking. The aim of this study was to examine the impact of hypoxia and simulated reperfusion on the in vitro uptake and release of nanomedicines by MDA-MB-231 human breast cancer cells. Cells were exposed to a hypoxic preconditioning treatment in 1% oxygen for 6 and 24 hours to induce temporal changes in the hypoxic circuit (e.g. HIF-1α expression). The preconditioned cells were then dosed with nanoparticles for 45 or 180 minutes emulating nanomedicine access following tumor reperfusion. Pericellular oxygen monitoring indicated oxygen tension declined exponentially to 2.3% within 30 minutes and 1.3% within 60 minutes. Hypoxic preconditioning significantly increased nanoparticle retention by up to 10% when compared to normoxic cultures, with the greatest relative difference between normoxic and hypoxic cultures occurring with a 45 minute dosing interval. Exocytosis studies indicated that the preconditioned cells had a significantly increased nanoparticle efflux (up to 9%) when compared to normoxic cells. Overall, we were able to show that hypoxic preconditioning regulates both the endocytosis and exocytosis of nanomedicines in human breast cancer cells

Heparin modified polyethylene glycol microparticle aggregates for focal cancer chemotherapy

Focal cancer therapy can improve clinical outcomes. Here, we evaluated injectable heparin-containing hydrogel material loaded with doxorubicin as a focal breast cancer therapy. We utilized a binary heparin/polyethylene glycol (PEG) hydrogel that was processed post synthesis into hydrogel microparticle aggregates to yield a readily injectable hydrogel. When loaded with doxorubicin, the injectable hydrogel microparticle aggregates had excellent short- and long-term anticancer activity against human breast cancer cells in vitro. Efficacy as a focal anticancer therapy was also evaluated in vivo by local injection of the doxorubicin-loaded PEG-heparin hydrogel microparticle aggregates into mice with established human orthotopic breast tumours. Animals showed significant antitumour responses by reduction in both primary tumour growth and metastasis when compared to animals which received the equivalent doxorubicin dose via an intravenous bolus injection. Overall, PEG-heparin hydrogel microparticle aggregates are emerging as a potential anticancer drug delivery system for focal therapy