Non-Amphiphilic Antimicrobial Polymers

Abstract

Antimicrobial resistance (AMR) is a severe threat to modern healthcare and must be addressed to prevent millions of deaths in the coming decades. Antimicrobial polymers (APs) do not provoke resistance and are promising alternatives to conventional antibiotics. Classic APs possess an amphiphilic structure (cationic and hydrophobic). Herein, we question the necessity of amphiphilicity in APs and find that hydrophobicity is not an essential quality in these polymers. Combining cationic monomers with hydrophilic subunits containing hydrogen bond donors results in excellent antibacterial activity and concurrently low unspecific toxicity. Non-amphiphilic APs have the unique ability to cluster in isolated membrane regions, creating a supramolecular multivalence that enhances their membrane activity and aggregates bacterial cells. This effect, which only unfolds in the absence of hydrophobicity, opens new possibilities in the design of antimicrobial materials.

Keywords: Amphiphilicity; Antimicrobial polymers; Hydrogen bonding; Membrane interaction; RAFT polymerization.

Figure 1

a) Schematic overview of synthesized polymers with varying comonomer composition (30%, 50%, and 70% of noncationic comonomer). A mixture of CTAs (grey) was used for PI‐RAFT polymerization. b) Determination of hydrophobicity and amphiphilicity of polymer via HPLC (scatter) and surface tension (bars) measurements, respectively. HPLC was performed using a water/acetonitrile gradient and retention time (peak maximum) is plotted as scatter plot with error range representing the peak width at half height. Surface tension was determined via a Wilhelmy‐plate and bars represent maximum change in water surface tension upon polymer addition. c) Determination of H‐bonding capacity via FTIR spectroscopy monitoring the C═O stretch vibration (spectra are overlayed with extracted values of peak maximum and center of mass). The method was not applicable to MA copolymers as here the C═O is part of an ester function that shows an intrinsically different wavenumber and thus cannot be compared to amides.

Figure 2

a) MIC₅₀ values against EC and PA as function of polymer composition. Performance plots against EC comparing selectivity (based on HC₁₀) and TI (based on CC₅₀) for polymers with systematically varied amphiphilicity b) or H‐bonding capacity c). The symbol size is linked to the surface activity of polymers with larger sizes indicating higher amphiphilicity of respective macromolecules.

Figure 3

a) Dye leakage study using liposomes from 2‐oleoyl‐1‐palmitoyl‐sn‐glycero‐3‐phosphoethanolamine (POPE) and 2‐oleoyl‐1‐palmitoyl‐sn‐glycero‐3‐phospho‐rac‐(1‐glycerol) (POPG) (8:2); EC₅₀ values are based on Hill1‐fit using Origin software. b) QCM‐D measurements (mass increase) of polymers (all 70% comonomer) on silicon sensors carrying a supported bilayer of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) and 1,2‐dioleoyl‐sn‐glycero‐3‐phospho‐l‐serine (DOPS) (9:1). Polymers were added at t = 0. c) Raman spectra of mixtures of polymers with POPE in a 1:2 mixture. Peaks 1 and 2 are associated with the aliphatic region of the lipid. d) Interaction factor based on the ratio of Raman peaks 2/1 as a function of the respective comonomer.

Figure 4

a) Polymer binding to GUVs as quantified by evaluating the total fluorescence intensity emitted by polymer molecules colocalized with lipid membranes in each image, normalized by the amount of pixels belonging to GUVs (see Methods for details, total data amount of points: 157), b) FRET intensity of polymers in contact with bi‐lipid membrane (EC mimic) in the form of liposomes as a function of time, c) microscopic images of labeled GUVs mimicking EC membranes and labeled polymers. Images were contrast enhanced to increase visibility; scale bars are 50 µm.

Figure 5

Confocal microscopy images of bacteria treated with dye‐labeled copolymers. Bacteria were incubated with either PBS (“No Polymer”) or a 20 nM solution of Alexa405‐labeled polymer for 1 h, shortly centrifuged and resuspended in Luria/Bertani medium before imaging. Zoomed images (bottom row) were acquired in samples additionally containing 0.5% agarose to minimize the lateral movements of the bacteria. All images were acquired at room temperature (23 ± 1 °C). Scale bars are 20 µm (2 µm for zoomed pictures).

Scheme 1

Sketch of differences in membrane interaction between amphiphilic and non‐amphiphilic APs.