A Silk-Based Platform to Stabilize Phenylalanine Ammonia-lyase for Orally Administered Enzyme Replacement Therapy

Abstract

Phenylalanine ammonia-lyase (PAL) has gained attention in recent years for the treatment of phenylketonuria (PKU), a genetic disorder that affects ~1 in 15,000 individuals globally. However, the enzyme is easily degraded by proteases, unstable at room temperature, and is currently administered in PKU patients as daily subcutaneous injections. We report here the stabilization of the PAL from Anabaena variabilis, which is currently used to formulate pegvaliase, through incorporation in a silk fibroin matrix. The combination with silk stabilizes PAL at 37 C. In addition, in vitro studies showed that inclusion in a silk matrix preserves the biological activity of the enzyme in simulated intestinal fluid, enabling the oral administration of pegvaliase for the treatment of PKU.

Reshaping de Novo Protein Switches into Bioresponsive Materials for Biomarker, Toxin, and Viral Detection

“…Silk Fibroin ISOLATION: Regenerated silk fibroin (RSF) solution was isolated from B. mori cocoons as previously reported. [27] Briefly, silk cocoons were shredded, separated from impurities, and boiled in a 0.02 m solution of sodium carbonate to remove sericin. Boiling time was set to 120 min to fabricate bio-reactive films, sponges, and inks, and to 30 minutes to prepare the mix for electrospinning.…”

“…[22][23][24][25][26] Additionally, the amphiphilic nature of silk protein chains allows for the generation of pico-to nanoliter solution drops of silk, enabling use in printing technologies without modifying the rheological properties of the inks (in terms of viscosity and surface tension) with non-compatible organic or inorganic surfactants, which could negatively interact with the doping agents, therefore enabling the functionalization of any substrate of interest. [22] Furthermore, due to its structure, RSF can stabilize intrinsically labile biomolecules, such as drugs, [27][28][29] enzymes, [30][31][32] pH indicators, [33] and complex biofluids, [34] thereby resulting in a promising matrix for the development of shelf-stable protein-based biosensors.…”

Boronic Acid-Tethered Silk Fibroin for pH-Dependent Mucoadhesion

“…Regenerated silk fibroin solution was isolated from B. mori cocoons following a method previously reported in the literature . Briefly, silk cocoons were shredded, manually separated from impurities, and boiled in a 0.02 M solution of sodium carbonate for 120 min to remove the sericin that binds the fibroin fibers together.…”

“…Regenerated silk fibroin solution was isolated from B. mori cocoons following a method previously reported in the literature. 26 Briefly, silk cocoons were shredded, manually separated from impurities, and boiled in a 0.02 M solution of sodium carbonate for 120 min to remove the sericin that binds the fibroin fibers together. The fibers were rinsed with deionized water three times for 20 min and air-dried for over 12 h. The dried silk mats were subsequently dissolved in a 9.3 M solution of lithium bromide at 60 °C for 4 h. The chaotropic salt was then removed through dialysis using membranes with a MWCO of 3.5 kDa against deionized water (DI) for 48 h, changing the water 6 times at regular intervals.…”

“…Regenerated silk fibroin (silk), produced from Bombyx mori silkworms, is a promising natural polymeric matrix that can be processed into a variety of functional material formats, including sponges, films, hydrogels, and microneedles. − Silk is biodegradable, biocompatible, and safe for vaginal administration . In the present work, silk was chemically modified with boronic acid pendant groups to increase its mucoadhesive properties at different pH values.…”