Evaluation of the Antimicrobial Activity in Host-Mimicking Media and In Vivo Toxicity of Antimicrobial Polymers as Functional Mimics of AMPs

Activity tests for synthetic antimicrobial compounds are often limited to the minimal inhibitory concentration assay using standard media and bacterial strains. In this study, a family of acrylamide copolymers that act as synthetic mimics of antimicrobial peptides were synthesized and shown to have a disruptive effect on bacterial membranes and structural integrity through microscopy techniques and membrane polarization experiments. The polymers were tested for their antimicrobial properties using media that mimic clinically relevant conditions. Additionally, their activity was compared in two different strains of the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Pseudomonas aeruginosa. We showed that the medium composition can have an important influence on the polymer activity as there was a considerable reduction in minimal inhibitory concentrations against S. aureus grown in synthetic wound fluid (SWF), and against P. aeruginosa grown in synthetic cystic fibrosis sputum media (SCFM), compared to the concentrations in standard testing media. In contrast, we observed a complete loss of activity against P. aeruginosa in the serum-containing SWF. Finally, we made use of an emerging invertebrate in vivo model, using Galleria mellonella larvae, to assess toxicity of the polymeric antimicrobials, showing a good correlation with cell line toxicity measurements and demonstrating its potential in the evaluation of novel antimicrobial materials.


Synthesis of Boc-AEAM
Boc-AEAM was synthesised according to the literature. In two steps as summarised below. 2 Step 1: Synthesis of N-t-butoxycarbonyl-1,2-diaminoethane Ethylenediamine (4.41 g, 4.9 mL, 73 mmol) was dissolved in 40 mL of DCM and then transferred into a 2necked 100 mL flask equipped with a condenser, a pressure equalising dropping funnel and a nitrogen inlet. After cooling down the reaction mixture with an icebath, a solution of Boc-anhydride (3.98 g, 18 mmol) in DCM (20 mL) was added dropwise over 2 hours with stirring.
Subsequently, the reaction mixture was allowed to warm up to RT and stirred overnight. The solvent was then removed by rotary evaporation and a precipitate, identified as N,N'-(bis-tbutoxycarbonyl)-1,2-diaminoethane, was observed upon addition of water (50 mL). After filtration, the resulting product was saturated with NaCl and extracted with EtOAc (3 x 60 mL).
The combined organic phases were concentrated under reduced pressure resulting in a pale oil.
Residual NaCl was removed by dissolution of the oil in CHCl 3 and filtration of the solution. The solvent was removed under reduced pressure to give a colourless oil identified as N-tbutoxycarbonyl-1,2-diaminoethane (1.51 g, 9 mmol, 50 %).

Step 2: Synthesis of N-t-butoxycarbonyl-N'-acryloyl-1,2-diaminoethane
A solution of acryloyl chloride (0.67 g, 0.6 mL, 7.4 mmol) in CHCl 3 (30 mL) was cooled down in an ice bath. Subsequently, a solution of NEt 3 (0.63 g, 0.9 mL, 6.2 mmol) and N-t-butoxycarbonyl-1,2-diaminoethane (1 g, 6.2 mmol) in CHCl 3 (15 mL) was added dropwise to the reaction mixture over a period of an hour and a half. The reaction mixture was then allowed to warm up to RT and stirred for an hour before the solvent was removed under reduced pressure.
The residue was washed with water (20 mL) and extracted with CHCl 3 (3 x 20 mL). The organic fractions were collected, combined and the solvent was removed under vacuum to obtain

Diblock and triblock copolymer synthesis by RAFT polymerisation
The following general procedure was used for all RAFT polymerisations.

Synthesis of the first block
Monomer, initiator (VA-044), CTA (PABTC) and solvents (80% dioxane, 20 % water) were introduced in a vial with a magnetic stirrer and a rubber septum. The solution was degassed with nitrogen approximately for 20 min. Then, the reaction vial was placed in an oil bath at 46 °C for 6 hours to perform the RAFT polymerization. After 6h, the test tube was withdrawn from the oil bath and a sample was taken for 1H NMR ( Figure S3) and GPC analysis. ( Figure S9).

Synthesis of subsequent blocks
The reaction vial with the reaction mixture was opened and additional monomer, initiator and solvent were introduced. The reaction vial was sealed with a rubber septum and degassed with nitrogen approximately for 20 min. Then, the reaction vial was placed in an oil bath at 46 °C for 6 hours to perform the RAFT polymerization. After 6 h, the test tube was withdrawn from the oil bath and a sample was taken for 1H NMR ( Figure S4-8) and GPC analysis ( Figure S9, Table S2).
The quantity of reagents needed for the diblock and triblock copolymers is summarized in Table   S1.

Calculation of M n,th
The theoretical number average molar mass (M n,th ) was calculated as were determined by comparison with poly(methyl methacrylate) standards (Agileny EasyVials) using using Agilent GPC/SEC software. GPC traces are shown in Figure S9 and dispersity (Đ) in Table S2.  GmbH) (9.29 mg, x 14.21 μmol, 1.5 eq.) was added to the polymer solution and stirred overnight in the dark ( Figure S10). Figure S10. Reaction scheme of polymer-Cy5 conjugation by orthogonal chemistry.
The sample was purified by precipitation in ether and boc groups were deprotected and polymer purification was carried out as described in the next section. Purity was checked by HPLC ( Figure   S11). Deprotection of the polymers TFA was added directly to the polymeric solution in DCM, stirred for 3 hours at 40 °C. After the reaction took place, TFA was removed by precipitation in cold diethyl ether three times. In order to replace the TFA counter-ion, the polymers were dialyzed against a NaCl solution, followed by dialysis against distilled water for 3-4 days. Boc-group removal was monitored by 1H NMR and 19F NMR used to monitor for traces of the TFA counterion. (Figures S12-15). Finally, the dialyzed product was freeze-dried and stored at 4 ℃.    where σ is the standard deviation, and d is the diameter, both obtained from the number-weighted particle size distribution.  The power law component is described by the exponent , and scaling factor , proportional to both the radius of gyration of the aggregates (described at inaccessibly low ) and the proportion of polymer chains undergoing aggregation. Finally, a constant background is summed into the model to account for the incoherent background. The Zero-angle intensity, I 0 , describes the intensity of the polydisperse Gaussian coil form factor in the absence of any Porod scattering contribution. The polydispersity, Ð, was fixed for each polymer throughout the fitting procedure based on values obtained though SEC analyses.  Figure S18. Haemolysis of sheep RBC in the presence of 1 mg mL -1 of polymeric materials.     . Scanning electron micrographs of S. aureus Newman exposed to g-D50 (MIC concentration) and P. aeruginosa PA14 exposed to a-T100-1 (MIC concentration).