3D-Printable and Enzymatically Active Composite Materials Based on Hydrogel-Filled High Internal Phase Emulsions

Abstract

The immobilization of enzymes in biocatalytic flow reactors is a common strategy to increase enzyme reusability and improve biocatalytic performance. Extrusion-based 3D bioprinting has recently emerged as a versatile tool for the fabrication of perfusable hydrogel grids containing entrapped enzymes for the use in such reactors. This study demonstrates the suitability of water-in-oil high internal phase emulsions (HIPEs) as 3D-printable bioinks for the fabrication of composite materials with a porous polymeric scaffold (polyHIPE) filled with enzyme-laden hydrogel. The prepared HIPEs exhibited excellent printability and are shown to be suitable for the printing of complex three-dimensional structures without the need for sacrificial support material. An automated activity assay method for the systematic screening of different material compositions in small-scale batch experiments is presented. The monomer mass fraction in the aqueous phase and the thickness of printed objects were found to be the most important parameters determining the apparent activity of the immobilized enzyme. Mass transfer limitations and enzyme inactivation were identified as probable factors reducing the apparent activity. The presented HIPE-based bioinks enable the fabrication of flow-optimized and more efficient biocatalytic reactors while the automated activity assay method allows the rapid screening of materials to optimize the biocatalytic efficiency further without time-consuming flow-through experiments involving whole printed reactors.

Immobilization of β-Galactosidase by Encapsulation of Enzyme-Conjugated Polymer Nanoparticles Inside Hydrogel Microparticles

“…The observed relative activity of H P E was higher than the activity of H P ENPs ( Figure 6A ), although the enzyme concentration was adjusted to result in the same activity for both microparticle samples. Leaching of the unbound enzyme may be a potential explanation for the observation of higher activity in H P E. Due to its small size, the unbound enzyme may be able to diffuse out of the hydrogel microparticles and exert higher activity in solution due to the reduced mass transfer limitations ( Wenger et al, 2020 ). Although a highly probable explanation to the observed activity change, the study of leaching was not in the scope of this paper.…”

“…Kinetic parameters can be evaluated using integrated reaction rate equations ( Goldstein, 1976 ; Carrara and Rubiolo, 1996 ). In a previous study, we have already investigated the kinetics of β-galactosidase immobilized in 3D-printed composite hydrogels based on high internal phase emulsions ( Wenger et al, 2020 ).…”

Enzyme entrapment, biocatalyst immobilization without covalent attachment

“…178 β-Galactosidase (β-Gal) from Aspergillus oryzae in water-inoil high internal phase emulsions (HIPEs) was used as a 3D printable bio-ink for enzyme entrapment in a cylinder shape hydrogel surrounded by a porous polymeric material. 179 The water-in-oil emulsions were made by mixing an oil phase containing of 2-ethylhexyl acrylate (EHA), isobornyl acrylate (IBOA), trimethylolpropane triacrylate (TMPTA), surfactant Pluronic® L-121, and photo initiator (Darocur®TPO) with a water phase, made from acrylic acid (AA), poly(ethyleneglycol) diacrylate (PEG-DA700), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and β-galactosidase. The emulsion was molded in a 48 cylindrical well microplate and subjected to UV-LED for polymerization at 25mW cm −2 for 2 min to give a hydrogel surrounded by an outer porous polymeric material.…”

Considerations Using Additive Manufacture of Emulsion Inks to Produce Respiratory Protective Filters Against Viral Respiratory Tract Infections Such as the COVID-19 Virus

“…This is expected as the emulsion is curing against air; therefore, there is no surface contact with a material that can adversely affect the surface porosity by surface destabilization. This technique has also been used to create a composite polyHIPE for use as a biocatalytic flow reactor using an enzyme-laden hydrogel as the emulsion droplet phase[ 104 ]. Alternatively, an emulsion can simply be injected directly into a void before bulk polymerization[ 25 ].…”

“…Extrusion-based printing of emulsion has been demonstrated to print at a speed of 10 mm s −1 with extrusion width of 0.6 mm using a modified RepRap style 3D printer[ 93 ]. Modified extrusion-based 3D printers have been used to print emulsions that are cured on demand, with print speeds tested up to 9 mm/s and layer heights of 100–300 μm[ 104 ].…”