Development and performance of a 3D‐printable poly(ethylene glycol) diacrylate hydrogel suitable for enzyme entrapment and long‐term biocatalytic applications

Abstract

Physical entrapment of enzymes within a porous matrix is a fast and gentle process to immobilize biocatalysts to enable their recycling and long‐term use. This study introduces the development of a biocompatible 3D‐printing material suitable for enzyme entrapment, while having good rheological and UV‐hardening properties. Three different viscosity‐enhancing additives have been tested in combination with a poly(ethylene glycol) diacrylate‐based hydrogel system. The addition of polyxanthan or hectorite clay particles results in hydrogels that degrade over hours or days, releasing entrapped compounds. In contrast, the addition of nanometer‐sized silicate particles ensures processability while preventing disintegration of the hydrogel. Lattice structures with a total height of 6 mm consisting of 40 layers were 3D‐printed with all materials and characterized by image analysis. Rheological measurements identified a shear stress window of 200 < τ < 500 Pa at shear rates of 25 s−1 and 25°C for well‐defined geometries with an extrusion‐based printhead. Enzymes immobilized in these long‐term stable hydrogel structures retained an effective activity of approximately 10% compared to the free enzyme in solution. It could be shown that the reduction of effective activity is not caused by a significant reduction of the intrinsic enzyme activity but by mass transfer limitations within the printed hydrogel structures.

Enzyme Scaffolds with Hierarchically Defined Properties via 3D Jet Writing

“…UV-vis spectra of the solutions were measured at 420 nm to monitor the absorption profile and intensity of the oNP product. [22] Using a commercially available oNP, a calibration curve was determined to consequently quantify the concentration. As the effective extinction coefficient may change in presence of solutes and dispersed particles, hydrogel scaffolds without enzymes were tested as a reference.…”

“…The diffusion coefficient and the Thiele modulus of around 5 are lower compared to other PEGDA hydrogels, which is due to the highmolecular weight PAA content and the lack of swelling. [22,34,35] The simulated diffusion of oNPG into a scaffold with a crosssectional area of 2.2 × 10 3 µm 2 is shown in Figure 6a. The reactant oNPG diffuses into the scaffold over time and is converted into oNP by the enzymes, leading to a steady-state concentration after 1 h. The oNP molecule does not undergo any further enzymatic transformation, in contrast to the reactant, and can diffuse further inside the scaffold (Figure 6b+d).…”

“…[21] Although extrusion-based printing can proceed under mild process conditions, they typically are limited by low resolution caused by the nozzle diameter of the printer and the applied pressure. [4,22] Electrospinning is a method that allows the production of thin polymer fibers in the micro-and nanometer range with a large spectrum of different polymers and was already used for cell encapsulation. [23,24] Unfortunately, random deposition of the fibers on the collector due to bending and whipping instabilities based on the applied electric field is common.…”

Simulative Minimization of Mass Transfer Limitations Within Hydrogel-Based 3D-Printed Enzyme Carriers

“…The simulation is based on experimental data for the immobilization of β-Galactosidase from Aspergillus oryzae in 3D-printed hydrogel lattices, which were already published by our group (Schmieg et al, 2019). In short, the hydrogel consists of a heterogeneous mixture of polyethylene-glycol diacrylate polymer chains as well as colloidal silicate particles and enzymes (Schmieg et al, 2018). It was 3D-printed with an extrusion printing system into rectangular hydrogel lattices ( Figures 1A,B) with outer dimensions of 13 × 13 × 3 mm.…”

“…For the optimization of processes conducted in such a 3D-printed fixed-bed reactor, this manuscript shows a simulative approach to find optimal geometric parameters for the given enzymatic kinetics of β-Galactosidase and the diffusional behavior of the used polyethylene glycol diacrylatebased hydrogel (Schmieg et al, 2018). Boundary conditions for experimental optimization are the limitations of the extrusion-based 3D-printer, for example the applicability of the printed material for extrusion.…”