Supramolecular self-associating amphiphiles (SSAs) as enhancers of antimicrobial agents towards Escherichia coli (E. coli)

Supramolecular self-associating amphiphiles (SSAs) are a class of amphiphilic salt which have demonstrated antimicrobial activity against both Gram-positive and Gram-negative bacteria. Herein, we show that SSAs are also able to increase the efficacy of a range of currently used antimicrobial/therapeutic agents with a range of different chemical structures and modes of antimicrobial action against Gram-negative Escherichia coli, which include: octenidine (an antiseptic); ampicillin (an antibiotic); and cisplatin (a DNA chelating agent). Additionally, we show these effects to be dependent on the order of agent addition. Finally, through completion of a range of 1 : 1 SSA :  antimicrobial/therapeutic agent physicochemical studies we gain an understanding as to how the self-association events and resultant SSA aggregate structure are effected by the presence of these secondary molecular species.


Experimental
General remarks: A positive pressure of nitrogen and oven dried glassware were used for all reactions. All solvents and starting materials were purchased from known chemical suppliers or available stores and used without any further purification unless specifically stipulated. The NMR spectra were obtained using a Burker AV2 400 MHz or AVNEO 400 MHz spectrometer. The data was processed using ACD Labs. NMR Chemical shift values are reported in parts per million (ppm) and calibrated to the centre of the residual solvent peak set (s = singlet, br = broad, d = doublet, t = triplet, q = quartet, m = multiplet). Tensiometry measurements were undertaken using the Biolin Scientific Theta Attension optical tensiometer. The data was processed using Biolin OneAttension software. A Hamilton (309) syringe was used for the measurements. The melting point for each compound was measured using Stuart SMP10 melting point apparatus. DLS and Zeta Potential studies were carried out using Anton Paar Litesizer TM 500 and processed using KalliopeTM Professional. Cellular growth curve measurements obtained using Thermo Scientific Multiscan Go 1510-0318C plate reader and recorded using the SkanIt Software 4.0 and a Clariostar plater reader using MARS data analysis software.
Tensiometry Studies: All the samples were prepared in an EtOH:H2O (1:19) solution. All samples underwent an annealing process in which the various solutions were heated to approximately 40 °C before being allowed to cool to room temperature, allowing each sample to reach a thermodynamic minimum. All samples were prepared through serial dilution of the most concentrated sample. Three surface tension measurements were obtained for each sample at a given concentration, using the pendant drop method. The average values were then used to calculate the critical micelle concentration (CMC).

DLS Studies:
All vials used for preparing the samples were clean and dry. All solvents used were filtered to remove any particulates that may interfere with the results obtained. Samples of differing concentrations were obtained through serial dilution of a concentrated solution. All samples underwent an annealing process, in which they were heated to 40 °C before being allowed to cool to 25 °C. A series of 9 runs were recorded at 25 °C. Zeta Potential Studies: All vials used for preparing the samples were clean and dry. All solvents used were filtered to remove any particulates that may interfere with the results obtained. All samples underwent an annealing process in which the various solutions were heated to approximately 40 °C before cooling to room temperature, allowing each sample to reach a thermodynamic minimum. The final zeta potential value given is an average of the number of experiments conducted at 25 °C.

Chemical synthesis
Compound 1: This compound was synthesised in line with our previously published methods. Proton NMR was found to match our previously published values. 4          Table S1 -Overview of diffusion coefficients (m 2 /s) for compound 1 only and co-formulations a-d in DMSO-d6 at 298 K. Errors for diffusion constants are no greater than ± 1 x 10 -13 m 2 /s.

Coformulation
65 83 a a = could not be determined due to peak overlap.

Self-association constant calculation
Co-formulation a -Dilution study in DMSO-d 6 0. 5                                Overview Table 5 -Overview of average DLS intensity particle size distribution peak maxima, zeta potential and CMC, measurements obtained for a H2O/ 5.0 % EtOH solution of an SSA (1) or co-formulation a-e (5.56 mM) at 298 K.* = experiments were performed at 0.56 mM due to solubility issues. Single Crystal X-ray structures  Table S6 -Hydrogen bond distances and angles observed for a single crystal sample produced from a solution of co-formulation a, calculated from the single crystal X-ray structure shown in Figure S73.      . Each coformulated system was studied where the: i) SSA was pre-incubated with the E. coli for 10 mins before the antimicrobial agent was added (green); ii) antimicrobial agent was pre-incubated with the E. coli for 10 mins before the SSA was added (orange); iii) SSA and antimicrobial agent was added in coformulation without any prior incubation (blue). The concentration of 2-6 alone was found to impede bacterial growth by < 30 % over 1100 mins.