Three-Dimensional Printable Enzymatically Active Plastics

Here, we describe a facile route to the synthesis of enzymatically active highly fabricable plastics, where the enzyme is an intrinsic component of the material. This is facilitated by the formation of an electrostatically stabilized enzyme–polymer surfactant nanoconstruct, which, after lyophilization and melting, affords stable macromolecular dispersions in a wide range of organic solvents. A selection of plastics can then be co-dissolved in the dispersions, which provides a route to bespoke 3D enzyme plastic nanocomposite structures using a wide range of fabrication techniques, including melt electrowriting, casting, and piston-driven 3D printing. The resulting constructs comprising active phosphotriesterase (arPTE) readily detoxify organophosphates with persistent activity over repeated cycles and for long time periods. Moreover, we show that the protein guest molecules, such as arPTE or sfGFP, increase the compressive Young’s modulus of the plastics and that the identity of the biomolecule influences the nanomorphology and mechanical properties of the resulting materials. Overall, we demonstrate that these biologically active nanocomposite plastics are compatible with state-of-the-art 3D fabrication techniques and that the methodology could be readily applied to produce robust and on-demand smart nanomaterial structures.

chloride and 50 mg carbenicillin at 25°C for 36-48 hours. The bacteria were then harvested through 23 centrifugation and either purified immediately or the pellets were stored at −20°C for future 24 purification. sfGFP (in the pBAD vector) was into BL21(DE3) E. coli cells and in Terrific Broth (TB; Fisher, 25 UK) supplemented with 50 mg carbenicillin at 37°C expressed with arabinose induction at O.D = 0.6. 26 The bacteria were then harvested 4 h after induction through centrifugation and either purified 27 immediately or the pellets were stored at −20°C for future purification. 28

Purification of arPTE 29
A bacterial cell pellet (from 500 mL of culture) was resuspended in 25 mL of buffer A (30 mM HEPES, 30 100 µM cobalt chloride, pH 8.0) and lysed through sonication. The lysate was clarified through high-31 speed centrifugation and the supernatant was retained. The supernatant was loaded onto a DEAE-32 Sepharose column (GE Healthcare, UK) that had been pre-equilibrated with buffer A, and the column 33 underwent isocratic elution with buffer A. The fractions containing arPTE were pooled and then 34 concentrated into a small volume (5-10 mL), which was then loaded onto a pre-equilibrated Superdex 35 200 pg HiLoad 26/600 column (GE Healthcare) and underwent isocratic elution with buffer A. The peak 36 fractions from the size exclusion chromatography (SEC) purification were pooled and was stored at 37 4°C for future use. The protein after SEC had single band purity as assessed by SDS-PAGE 38 electrophoresis. 39

Purification of sfGFP 40
A bacterial cell pellet (from 1 L) was resuspended in 30 mL of buffer B (30 mM HEPES, 200 mM NaCl, 41 20 mM imidazole, pH 8.0) and lysed through sonication. The lysate was clarified through high-speed 42 Supporting Information S3 centrifugation and the supernatant was retained. The supernatant was loaded onto a nickel affinity 43 column 10-15× column volumes of buffer B. The protein was then eluted with buffer C (30 mM HEPES, 44 200 mM NaCl, 250 mM imidazole, pH 8.0) and the fractions containing the protein were retained. The 45 protein was then dialyzed against buffer D (30 mM HEPES, 200 mM NaCl, pH 8.0). The proteins had 46 single band purity as assessed by SDS-PAGE electrophoresis. 47

Synthesis of oxidized IGEPAL-890 48
The method for IGEPAL-890 oxidation was followed as previously described by Armstrong et al. 1 In 49 brief, IGEPAL-890 (2 g, 1.0 mmol) was dissolved in MilliQ water (50 mL), and Sodium Bromide (50 mg, 50 0.49 mmol), Sodium Hypochlorite (5 mL, 10-15% available chlorine) and TEMPO (30 mg, 0.19 mmol) 51 was then added to the stirring solution. The solution was basified to pH 11.0 and allowed to stir 52 overnight, after which, the solution was acidified to pH 1.0 and extracted 3 times with chloroform (80 53 mL each). The chloroform was then washed 3 times with pH 1.0 MilliQ water (80 mL each) and then 54 the chloroform was removed by rotary evaporation. The resulting oil was further purified through 55 recrystallization in neat ethanol at −20°C. 56 Enzyme conjugation and melt preparation procedure 57 The amount of surfactant used for conjugation was determined using the following equation: 58 Moles of surfactant = Moles of enzyme × number of charged residues × 2.5 (eq. 1) 59 With the number of charged residues being the number of anionic residues for the cationic 60 surfactant, and the number of cationic residues for the anionic surfactant. The cationic surfactant 61 (Ethoquad HT 25) was dissolved in the appropriate buffer (A, D, or E. 30 mg/mL) and then rapidly 62 added to a stirring solution of enzyme (100-150 µM). The solution was stirred for 3 hours at which 63 point a solution containing anionic surfactant (oxidized IGEPAL-890), which was prepared in an 64 identical manner to the cationic surfactant, was added and the resulting solution allowed to stir for 3 65 hours or overnight. The solution then underwent dialysis with MilliQ water for 16-24 hours with one 66 Supporting Information S4 change of the external dialysis solution at 3 hours. The solution was then lyophilized, and the resulting 67 powder was thermally annealed (45°C) to create the enzyme melt. In this liquid form, the melt can be 68 dissolved in warm organic solvents (45°C). The presence of water, either due to ambient exposure or 69 inadequate lyophilization, can prevent the melt from correctly dissolving in organic solvents and can 70 result in turbidity. 71

Synchrotron Radiation Wide Angle X-ray Scattering (SR-WAXS) analysis of melt 72
The data for the [arPTE][S + ][S − ] melt was obtained using synchrotron radiation-wide angle X-ray 73 scattering (SR-WAXS) on the I22 beamline at the Diamond Light Source, Oxford. Samples were loaded 74 into punctured DSC pans lined with capton film, and 1 frame was recorded for 1 second per 1°C, 75 heating at 1°C a minute. Data preparation and reduction to generate the 2D scattering profiles were 76 performed using DAWN. 77

Synchrotron Radiation-Circular Dichroism (SR-CD) analysis of melt 78
The data for the [arPTE][S + ][S − ] melt was obtained using synchrotron radiation-circular dichroism 79 (SR-CD) B23 beamline at the Diamond Light Source. A thin film of the melt was held between two 80 quartz slides, and the CD spectrum was collected from 180-260 nm with one spectrum collected per 81 3°C, heating at 1°C a minute. 82

Piston-Driven 3D (PD3D) printing 83
Piston-driven 3D printing consists in the extrusion of a wide range of acellular viscous inks or 84 hydrogel from the nozzle of a syringe guided by a piston with an accurate resolution. The MendelMax 85 3.0 3D printer (Maker's Tool Works, US) was retrofitted with a custom extruder able to drive a 5 mL 86 syringe. The printing of the solution is driven by the motorized piston, composed by a NEMA17 stepper 87 motor connected with a metallic piston that moves the plunger of the syringe, ensuring a controlled 88 deposition of the polymeric solution. 89

S5
This approach allows the fabrication of the complex structure by a continuous deposition of the 90 material from the nozzle of the syringe to the stage. The 3-dimensionality of the structures is achieved 91 by a layer-by-layer extrusion of the polymeric solution, in which the deposition of the first layer is 92 followed by a period of stabilization to allow the evaporation of the volatile solvent, before the 93 extrusion of the second layer. 94 A low viscous solution of PCL was dissolved in high volatile solvent and printed through jetting and 95 extruding with high resolution as previously described. 2 In brief, PCL (average MW 80,000; Sigma 96 Aldrich) solution was prepared dissolving the polymer in chloroform at 20% (w/v) and the conjugated 97 enzymes were added at 1% (w/w). The solution of PCL-conjugated enzyme was then loaded into the 98 5 ml syringe and mounted on the extruder driven by the motorized piston. The simple geometry of a 99 ring was chosen to characterize the enzymatic activity in aqueous environment at different 100 timepoints. To test the correlation between the activity and physical features, 4 rings were fabricated 101 ( Figure S4): i) 1 layer, 21 rings; ii) 3 layers, 16 rings; iii) 5 layers, 11 rings; iv) 7 layers, 7 rings. The motion 102 speed of the piston was set-up at 0.2 mm•min −1 and a 27-gauge nozzle was used. 103 The feasibility of fabricating a PCL-conjugated enzyme structure with higher complexity was 104 successfully demonstrated by printing a woodpile. The structure consists in the overlap of 6 layers of 105 grid in order form a 3D matrix. For this experiment, a 30-gauge nozzle was used and the speed of the 106 piston 0.15 mm•min −1 was set-up achieving a line width of 100 µm. The use of our composite material 107 with this printing technique follows the same technical and structural capabilities and restrictions as 108 described previously. 2 109 Filament extrusion & thermal moulding procedure 110 PCL (average MW 80,000; Sigma Aldrich) was initially dissolved in chloroform and the conjugated 111 enzymes were added in the mixture at 1% w/w conjugate to PCL. The mixture was then left to dry for 112 the chloroform to completely evaporate overnight. The resulting enzyme-PCL hybrid material was 113 Supporting Information S6 processed into fine pieces and fed through a Noztek Pro extruder at 70°C and a filament of diameter 114 varying between 1.5-1.7 mm was generated. The filament was then used for thermal moulding of ring 115 specimens. An aluminium mould was used, around which the filament was wrapped to generate the 116 ring shape, which was kept at 60°C for 1 hour for the ring specimen to mould. The specimens were 117 left to cool for one hour and they were then removed from the mould for further testing. 118 Melt Electrowriting (MEW) procedure 119 PCL (average MW 45,000; Sigma Aldrich) was initially dissolved in chloroform and conjugated 120 enzyme or sfGFP were added in the mixture at 0.1% w/w and 0.2% w/w conjugate to PCL. The mixture 121 was then left to dry for the chloroform to completely evaporate overnight. The enzyme plastics were 122 loaded into a stainless-steel syringe of a melt electrowriting instrument (CAT000111, Spraybase), 123 which was equipped with a 24 G blunt-end spinneret. A gas pressure of 0.15 bar was supplied to 124 extrude the polymer melt out from the spinneret at 70°C, while the needle was located at 4 mm away 125 from the collector. A high voltage of 4 kV was applied between the collecting plate and the spinneret, 126 generating a strong electrostatic field that leads to the formation of Taylor  for environmental degradation, if any, of the PCL by water, and to ensure that the material was 144 constantly exposed to an environment where the enzyme could potentially be depleted.   Supporting Information S18 Conversely, we see that the melting transition is shifted asymmetrically towards a higher 271 temperature, although the peak melting transition does not change significantly (57.3 ± 0.1°C for 272 0.2% surfactant-PCL and 56.91 ± 0.01°C for 0.5% surfactant-PCL). As with the solvent-free enzyme 273 melts, all samples were analysed using aluminium non-hermetic pans. The temperature cycling 274 protocol for DSC analysis was as described in the methods.   into the PCL and the rate of cavitation/degradation. 1% enzyme-PCL (w/w) shows a greater degree 298 degradation when compared to 0.1% enzyme-PCL or enzyme-free PCL. Conversely, 0.1% enzyme-PCL 299 has a comparable level of degradation to the control enzyme, showing that at this level of enzyme 300 loading it should not affect the structural integrity of the material, at least in terms of environmental 301 degradation. It should be noted that the cavities shown in this figure in the 0.1% enzyme-PCL and the 302 control are only to serve as an example of cavitation, and that these cavities are otherwise rare and 303 are only sparsely observed for the overall sample. Samples were not coated for SEM analysis at any 304 stage and were analysed as described in the supporting information.