Oxygen-Tolerant RAFT Polymerization Initiated by Living Bacteria

Living organisms can synthesize a wide range of macromolecules from a small set of natural building blocks, yet there is potential for even greater materials diversity by exploiting biochemical processes to convert unnatural feedstocks into new abiotic polymers. Ultimately, the synthesis of these polymers in situ might aid the coupling of organisms with synthetic matrices, and the generation of biohybrids or engineered living materials. The key step in biohybrid materials preparation is to harness the relevant biological pathways to produce synthetic polymers with predictable molar masses and defined architectures under ambient conditions. Accordingly, we report an aqueous, oxygen-tolerant RAFT polymerization platform based on a modified Fenton reaction, which is initiated by Cupriavidus metallidurans CH34, a bacterial species with iron-reducing capabilities. We show the synthesis of a range of water-soluble polymers under normoxic conditions, with control over the molar mass distribution, and also the production of block copolymer nanoparticles via polymerization-induced self-assembly. Finally, we highlight the benefits of using a bacterial initiation system by recycling the cells for multiple polymerizations. Overall, our method represents a highly versatile approach to producing well-defined polymeric materials within a hybrid natural-synthetic polymerization platform and in engineered living materials with properties beyond those of biotic macromolecules.


Methods
Instrumentation 1 H NMR spectra were recorded at room temperature on a 400 MHz (Bruker DPX400 Ultrashield) using deuterated solvents (D 2 O). NMR spectra were analysed using MestReNova 11.0.0-17609 2016 Mestrelab Research S.L. DMF Size Exclusion Chromatography (SEC) was performed on Polymer Laboratories PL50+ system fitted with refractive index (RI) detector, two Agilent PLgel Mixed-D columns and a PLgel guard column eluted with DMF + 0.1% LiBr (w/w). Molecular weight (M n ) and polydispersity (Ð) were calculated according to PMMA narrow standards (2,200-0.8 kDa) using Agilent EasyVial calibrants fitted with a cubic function to correlate retention time and molar mass using Cirrus GPC software. Polymer samples were made by dissolving 3 mg/mL pure polymer in 1 mL DMF + 0.1% LiBr. 100 µL samples were injected and eluted at 1 ml/min for 30 min.
Aqueous SEC was performed on an Agilent 1200 system fitted with an RI and UV-vis detector set to 309 nm, Agilent two PL aquagel-OH column and one aquagel guard column eluted with 0.1 M NaNO 3 eluent. Molecular weight (M n ) and polydispersity (Ð) were calculated according to PEG narrow standards (1,500-0.105 kDa) using Agilent EasyVial calibrants fitted with a cubic function to correlate retention time and molar mass. Polymer samples were made by dissolving 3 mg/mL pure polymer in 0.1 M NaNO 3 . 50 µL samples were injected and eluted at 1 ml/min for 30 min.
Dynamic light scattering (DLS) measurements were performed using a Malvern Nano Zetasizer (Malvern Instruments, Malvern UK). Data was processed using dispersion technology software (v 7.13, Malvern instruments, Malvern UK) using the multiple narrow modes algorithm based upon a nonnegative least square fit to calculate the hydrodynamic radius and the polydispersity index (PD). The Zaverage is an intensity-based overall average hydrodynamic size based on a specific fit to the raw correlation function data. The PDI is a measure of the degree of polydispersity of the sample.
TEM analysis was carried out on a JEOL 2100Plus equipped with a Gatan Ultrascan 1000 XP camera. Nanoparticle samples were prepared at a concentration of 1 mg/mL in deionised water and spotted on grids (carbon film on a copper mesh) which were precleaned by a 5 second oxygen/argon plasma clean (Fischione model 1020 Plasma cleaner).

Experimental information
Bacterial culture C. metallidurans CH34 type strain was purchased from German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany (DSMZ 22839) and routinely grown by directly inoculating 5 mL LB media with the culture stock stored at -80°C. Cultures were grown aerobically, at 30°C in a shaking incubator at 200 rpm overnight (18 hours). Overnight grown cultures were used to inoculate 20 mL LB media to a starting OD 600nm = 0.1 and grown at 30°C shaking to an OD 600nm ~1. In preparation for the polymerisation, the cultures were centrifuged (6000 rpm, 10 minutes) and the pellets washed twice with PBS (10 mL) via centrifugation. Each of the washed pellets were re-suspended in 4 mL PBS. 1 mL (3.4x10 10 CFU ml -1 ) was added to the polymerisation reaction mixture (1 mL) to give total C. metallidurans count of ~3.4x10 10 in the 2 mL volume, or 1.7x10 10 CFU mL -1 (per mL). When comparing live and dead cultures, live cultures were stored on ice until used in polymerisation. Dead bacterial cultures were heat killed by incubating at 70°C for 20 minutes.

Toxicity of water-soluble monomers to C. metallidurans
Minimum inhibitory concentration (MIC) experiments of the monomers used in this work were carried out using microplate reader with 96-Well standard microplates (Costar 3363), measuring the OD600 nm every 30 minutes for up to 24 hours. An overnight bacterial culture was diluted to an OD600 0.01 and incubated at 37°C, 200 rpm for an additional 2 h. The culture was then diluted back to an OD600 of 0.005. Monomers were prepared at a range of concentrations through serial dilution. In 96-well plates 50 µl of cell culture was added to 50 µl of test compounds. Initial OD600 readings were taken using a 96-well plate reader (Infinite M Nano, TECAN), and plates were then left to incubate overnight; 24 h at 37°C. OD600 readings were taken every 30 min and MIC values were determined as the lowest concentration of test compound to inhibit growth; whereby OD600 value did not increase.

Bacterially initiated polymerisation procedure
Polymerisations were conducted with the following general procedure targeting DP400. DMA (100 mM), CTA (0.24 mM), Glu (100 mM), GOx (0.25 µM), Fe 3+ (7 µM) and 1 mL of bacteria were mixed together to a total volume of 4 mL and shaken overnight at 30 °C. To quench the polymerisation, the polymerisation mixture was centrifuged at 4000 g to pellet C.metallidurans cells and the supernatant was lyophilised. Conversions were assessed through 1 H NMR spectroscopy by dissolving the remaining powder in deuterium oxide for analysis. Variations to this procedure are noted below in Table S3. For kinetics experiments, polymerisations were scaled up to a total volume of 16 mL and shaken at 30 °C.
At varying timepoints, 4 mL aliquots were removed and treated as above. Polymerisations were also carried out in the absence of some components i) with bacteria but no FeCl 3 .6H 2 O; ii) without any bacteria; and iii) with no bacteria and no FeCl 3 .6H 2 O. Variations to this procedure are noted below in Table S3.
The pure polymer was isolated by lyophilization.

Recycling C. met culture in successive polymerisations
Polymerisations were set up as described above and shaken overnight at 30 °C. Following 24 hours of incubation, the suspensions were separated by centrifugation as above and the supernatant was collected for further analysis. The bacterial pellet was resuspended in 1 mL DPBS and another mixture of DMA, CTA, FeCl 3. 6H 2 O, GOx, and Glucose as above were added to the tube. This procedure was repeated so that three successive polymerisations were performed.