Design of Quorum Sensing Inhibitor–Polymer Conjugates to Penetrate Pseudomonas aeruginosa Biofilms

Antimicrobial resistance (AMR) is a global threat to public health with a forecast of a negative financial impact of one trillion dollars per annum, hence novel therapeutics are urgently needed. The resistance of many bacteria against current drugs is further augmented by the ability of these microbes to form biofilms where cells are encased in a slimy extracellular matrix and either adhered to a surface or forming cell aggregates. Biofilms form physiochemical barriers against the penetration of treatments such as small molecule antibacterials, rendering most treatments ineffective. Pseudomonas aeruginosa, a priority pathogen of immediate concern, controls biofilm formation through multiple layers of gene regulation pathways including quorum sensing (QS), a cell-to-cell signaling system. We have recently reported a series of inhibitors of the PqsR QS regulator from this organism that can potentiate the action of antibiotics. However, these QS inhibitors (QSIs) have shown modest effects on biofilms in contrast with planktonic cultures due to poor penetration through the biofilm matrix. To enhance the delivery of the inhibitors, a small library of polymers was designed as carriers of a specific QSI, with variations in the side chains to introduce either positively charged or neutral moieties to aid penetration into and through the P. aeruginosa biofilm. The synthesized polymers were evaluated in a series of assays to establish their effects on the inhibition of the Pqs QS system in P. aeruginosa, the levels of inhibitor release from polymers, and their impact on biofilm formation. A selected cationic polymer–QSI conjugate was found to penetrate effectively through biofilm layers and to release the QSI. When used in combination with ciprofloxacin, it enhanced the biofilm antimicrobial activity of this antibiotic compared to free QSI and ciprofloxacin under the same conditions.

Thermal initiator 4,4′-Azobis(4-cyanovaleric acid) (ACVA, >98%) was purchased from Sigma Aldrich and used as received. RAFT agent propionic acid n-butyl trithiocarbonate (PABTC) was synthesised as previously reported. 1 Solvents and other reagents were acquired from commercial sources and used as received unless stated otherwise.

Instrumentation and Analysis
NMR spectroscopy 1 H NMR and 13 C NMR spectra were recorded on a Bruker DPX-400 spectrometer using deuterated solvent (materials section).

Size exclusion chromatography
A Polymer Laboratories PL-50 instrument equipped with differential refractive index (DRI) was used for SEC analysis. The system was fitted with 2 x PLgel Mixed D columns (300 x 7.5 mm) and a PLgel 5 µm guard column. The eluent used was DMF with 0.1% LiBr. Samples were run at 1 min -1 at 50°C.
Poly(methyl methacrylate) standards (Agilent EasyVials) were used for calibration between 955,500 -550 g mol -1 . Analyte samples were filtered through a membrane with 0.22 μm pore size before injection.
Respectively, experimental molar mass (M n,SEC ) and dispersity (Đ) values of synthesised polymers were determined by conventional calibration using Cirrus GPC software.  To a stirred solution of QSI (1 g, 2.7 mmol) and 1-carboxyethyl acrylate (0.5 g, 3.5 mmol) in dichloromethane (70 mL), EDCI.HCl (0.54 g, 3.5 mmol) and DMAP (0.033 g, 0.27 mmol) were added and the resulting mixture was stirred for 16 h. Subsequently, the reaction mixture was washed with water and then washed with saturated sodium bicarbonate solution and the organic layer was dried over granular magnesium sulphate. The organic layer was then reduced, and the crude residue was purified using flash chromatography using Hexane:Ethyl Acetate 8:2 to afford the product as a white solid (0.74 g, 55%). 1

Polymer synthesis
Polymers were synthesised using the following procedure, utilising the conditions described in Table   S1. PABTC, monomers (in the appropriate ratios) and ACVA (from a pre-made stock solution in DMSO) were dissolved in DMSO. The solution was fitted with an appropriately sized rubber septum and purged with nitrogen for 20 min. The polymerisation solution was subsequently immersed in an oil bath preheated to 70°C until the polymerisation reached 80-90% monomer conversion as determined by 1 H NMR spectroscopy. The reaction was cooled to ambient temperature and opened to oxygen to quench the polymerisation. Polymers were diluted 3-fold in acetone and purified by precipitation twice in diethyl ether (20-fold volume). The precipitated polymer was dissolved in dichloromethane (DCM), dried under reduced pressure and residual solvents were removed in a vacuum oven at 40°C for 48 h. Fluorescent analogues were synthesised using the same conditions with 1 mol% of acryloxyethyl thiocarbamoyl rhodamine B relative to the total monomer concentration (~1 fluorescent dye per polymer chain).

Microbiological experiments Biosensor reporter assay:
To evaluate the activity of QSI-polymer conjugates, the reporter strain P. aeruginosa PAO1-L mCTX::P pqsA -lux was used as previously described. 2 For testing, the polymers were assessed at the specified concentrations prepared from 50 mM DMSO stocks.

Biofilm viability and polymer penetration studies
Mature 2-day-old PAO1-L biofilms were used to characterise the effect of P2-QSI polymer and ciprofloxacin treatment combinations. Biofilms were grown on round glass coverslips (13 mm Ø, #1.5 thickness) under dynamic conditions (20 rpm) in FAB 10 mM glucose medium with or without supplementation with P2-QSI (100 μM), inoculated with diluted (OD 600nm = 0.01) bacteria from overnight cultures in LB. The biofilms were cultivated at 30°C for 2 days with medium replacement after 24h incubation, then washed in PBS to remove loosely attached cells and incubated for a further 6 or 24 h in fresh medium supplemented with various treatments. These included free ciprofloxacin 60 μg/mL (x300 the MIC of planktonic P. aeruginosa cells) 3 , QSI at 10 μM and ciprofloxacin in combination with QSI. Biofilms exposed to each treatment were washed in PBS and the viability of attached cells was evaluated by fluorescent staining using the LIVE/DEAD® BacLight™ Bacterial Viability kit (Molecular Probes, Life Technologies) according to manufacturer instructions. Following staining, coverslips were rinsed with distilled water and imaged using a LSM700 AxioObserver (Carl Zeiss, Germany) confocal laser scanning microscope (CLSM). Viable and non-viable biofilm biomass quantification from image stacks of biofilms was done with Fiji-ImageJ software. Live/dead ratios were established for each treatment and compared to untreated controls.
In parallel, and to ascertain polymer diffusion through the biofilm matrix, a Rhodamine B labelled analogue of P2-QSI conjugate was also tested against 2-day-old PAO1-L biofilms. After 12 h of incubation with the fluorescent polymer at 50 μM, biofilm samples were collected and stained with Syto9 fluorescent dye prior to CLSM image acquisition to simultaneously detect bacterial cells (green fluorescence) and P2-QSI polymer (red fluorescence). P2-QSI diffusion was assessed by quantifying the Rhodamine signal associated with each image stack and normalised to the biofilm biomass at different depths.

Statistical analysis
Graphical representations and statistical analysis were performed in GraphPad Prism, version 8.
Statistical differences were analysed using either multiple t-tests adjusted for multiple comparisons or one-way ANOVA with multiple comparisons, with p<0.05 used to indicate significance.   Figure S3. Effect of QSI and QSI polymers on a P pqsA -lux transcriptional fusion in PAO1-L, which reports the PQS-dependent activation of the pqs operon mediated by PqsR. A) Comparison of interference with the PQS system in PAO1-L by polymers P1 and P2 at 50 µM and QSI at 10 µM. B) Inhibition of the pqs promoter (P pqsA ) activity in response to P2-QSI supplemented at 0.5 µM to 50 µM relative to QSI treatment. Values given are averages from three different cultures ± standard deviation and correspond to the relative light units normalised to culture density (RLU/OD 600 ) over time (24 h).