Degradation Behavior of Silk Nanoparticles—Enzyme Responsiveness

Abstract

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.

The Biomedical Use of Silk: Past, Present, Future

“…Silk fibroin sequence alignment indicates a susceptibility to a number of proteases (e.g., protease XIV, α‐chymotrypsin, proteinase K, papain, matrix metalloproteinases, collagenase, etc. )). Nonetheless, predicting silk fibroin degradation simply based on the primary sequence is unreliable; for example, chymotrypsin has 434 cleavage sites in the silk heavy chain and 81 in the light chain, while protease XIV has 348 in the heavy chain and 41 in the light chain.…”

“…Nonetheless, predicting silk fibroin degradation simply based on the primary sequence is unreliable; for example, chymotrypsin has 434 cleavage sites in the silk heavy chain and 81 in the light chain, while protease XIV has 348 in the heavy chain and 41 in the light chain. Despite numerous cleavage sites, chymotrypsin treatment for 20 days had no quantifiable effect on silk fibroin, while protease XIV significantly degraded silk fibroin in vitro . Papain, a cysteine protease enzyme that mimics the activity of lysosomal enzymes, has 26 cleavage sites in the silk heavy chain (albeit exclusively in the amorphous regions) and 15 in the light chain, and it caused significant silk fibroin degradation over 20 days but at a slower rate than protease XIV.…”

“…Papain, a cysteine protease enzyme that mimics the activity of lysosomal enzymes, has 26 cleavage sites in the silk heavy chain (albeit exclusively in the amorphous regions) and 15 in the light chain, and it caused significant silk fibroin degradation over 20 days but at a slower rate than protease XIV. Similar observations were made with isolated lysosomal enzyme preparations …”

“…The current working model supports the notion that, for Bombyx mori silk, degradation begins with the 11 hydrophilic amorphous segments in the silk heavy chain, as well as the C‐terminal and N‐terminal and the silk light chain, which consist of completely nonrepeating amino acid sequences; this is then followed by degradation of the more crystalline sequences. [51,61c,89] The tightly packed crystalline domains are degraded last . Furthermore, the silk format is a critical factor in determining degradation rates, as in vivo studies in rodent models indicated faster degradation for open silk structures than for tightly packed monolithic silk fibroin films (rank order: hydrogel > silk scaffold > monolithic film) …”

Microfluidic-assisted silk nanoparticle tuning

“…8,11 Both native and PEGylated silk nanoparticles have demonstrated high drug loading efficacy, pH-dependent drug release, and selective degradation by protease enzymes as well as by ex vivo lysosomal enzymes. 12 Silk nanoparticles can be manufactured by a broad spectrum of methods (reviewed in ref. 6 and 13), including poly(vinyl alcohol) blending (size range 300 nm to 10 mm), 14 emulsication (170 nm), 15 capillary microdot printing (25-140 nm), 16 salting out (486-1200 nm), 17 supercritical uid technologies (50-100 nm), 18 ionic liquid dissolution (180 nm), 19 electrospraying (59-80 nm), 20 vibrational splitting of a laminar jet (up to 400 mm), 21 electric elds (200 nm to 3 mm), 22 milling technologies (200 nm), 23 and organic solvent desolvation (35-170 nm).…”