Biomimetic and biopolymer-based enzyme encapsulation.

Encapsulated enzymes are stable under various conditions and used in enzyme therapy, catalysis, and biosensors. The capsules are often inspired by structures from nature such as viral capsids, DNA motifs and diatom frustules. They are based on inorganic minerals as well as soft or polymeric materials, or even a combination of these. The choice of material influences the enzyme loading and response to heat, pH and presence of proteases. This review provides a comparison of enzyme encapsulation based on these different principles with a focus on materials inspired by nature.

Biomimetic Silica Encapsulation of Lipid Nanodiscs and ß-Sheet-Stabilized Diacylglycerol Kinase.

Integral membrane proteins (IMPs) comprise highly important classes of proteins such as transporters, sensors, and channels, but their investigation and biotechnological application are complicated by the difficulty to stabilize them in solution. We set out to develop a biomimetic procedure to encapsulate functional integral membrane proteins in silica to facilitate their handling under otherwise detrimental conditions and thereby extend their applicability. To this end, we designed and expressed new fusion constructs of the membrane scaffold protein MSP with silica-precipitating peptides based on the R5 sequence from the diatom Cylindrotheca fusiformis. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) revealed that membrane lipid nanodiscs surrounded by our MSP variants fused to an R5 peptide, so-called nanodiscs, were formed. Exposing them to silicic acid led to silica-encapsulated nanodiscs, a new material for stabilizing membrane structures and a first step toward incorporating membrane proteins in such structures. In an alternative approach, four fusion constructs based on the amphiphilic ß-sheet peptide BP-1 and the R5 peptide were generated and successfully employed toward silica encapsulation of functional diacylglycerol kinase (DGK). Silica-encapsulated DGK was significantly more stable against protease exposure and incubation with simulated gastric fluid (SGF) and intestinal fluid (SIF).

Continuous Flow Reactors from Microfluidic Compartmentalization of Enzymes within Inorganic Microparticles.

Compartmentalization and selective transport of molecular species are key aspects of chemical transformations inside the cell. In an artificial setting, the immobilization of a wide range of enzymes onto surfaces is commonly used for controlling their functionality but such approaches can restrict their efficacy and expose them to degrading environmental conditions, thus reducing their activity. Here, we employ an approach based on droplet microfluidics to generate enzyme-containing microparticles that feature an inorganic silica shell that forms a semipermeable barrier. We show that this porous shell permits selective diffusion of the substrate and product while protecting the enzymes from degradation by proteinases and maintaining their functionality over multiple reaction cycles. We illustrate the power of this approach by synthesizing microparticles that can be employed to detect glucose levels through simultaneous encapsulation of two distinct enzymes that form a controlled reaction cascade. These results demonstrate a robust, accessible, and modular approach for the formation of microparticles containing active but protected enzymes for molecular sensing applications and potential novel diagnostic platforms.

Silica particles with a quercetin-R5 peptide conjugate are taken up into HT-29 cells and translocate into the nucleus.

Intracellular delivery of bioactive polyphenols is currently evaluated as a protective strategy for cells under pharmaceutical stress. To this end, the 20mer R5 peptide from the marine diatom C. fusiformis was N-terminally modified with a quercetin derivative. This polyphenol-peptide conjugate was used to generate homogeneous silica particles under biomimetic conditions that are efficiently taken up by eukaryotic cells without being cytotoxic. However, not only was accumulation in the cytoplasm of living cells observed via electron and fluorescence microscopy but also translocation into the nucleus. The latter was only seen when the quercetin-peptide conjugate was present within the silica particles and provides a novel targeting option for silica particles to nuclei.