Antibacterial and Potentiation Properties of Charge-Optimized Polyrotaxanes for Combating Opportunistic Bacteria

Abstract

Bacteria are now becoming more resistant to most conventional antibiotics. Approaches for the treatment of multidrug-resistant bacterial infections are urgently required. Cationic polymers have broad-spectrum antibacterial activity but can also induce non-specific damage to mammalian cells. Herein, we report on the design of cationic polyrotaxanes (cPRs) with variable charge densities. cPRs were prepared by conjugating neutral ethanolamine and cationic ethylenediamine at various ratios onto threaded alpha-cyclodextrins and their antimicrobial and cytocompatible properties were investigated in vitro. In contact with Gram-negative bacteria, cPRs can disrupt the bacterial outer membrane integrity via electrostatic interactions and penetrate into the cytosol. The ability of cPRs to serve as potentiators at sub-MIC concentrations, to enhance the permeability and activity of poorly permeable antibiotics such as vancomycin, erythromycin and rifampicin, was also investigated against Gram-negative P. aeruginosa PAO1 and E. coli ATCC 25922.

Fig. 1

TEM of cPR2 reveals ca. 20 nm crescent-shaped morphological structures (A), and hydrodynamic size of all cPRs prepared averaged ca. 15 nm (B).

Fig. 2

LDH release comparing cPR1 and cPR2 in J77A4.1 mouse macrophage cells. cPR2 at +21 mV affects the mammalian cell plasma membrane significantly less than cPR1 characterized by a zeta potential of +36 mV at all concentrations investigated.

Fig.3

Bactericidal time-kill of cPR2 at 2x MIC against E. coli ATCC 25922, P. aeruginosa PAO1 and S. aureus ATCC 25923 reveal >99.9% bacteria are dead after 2–3 h incubation (A); error bars represent mean ± standard deviation for n = 3, *** p<0.001, ** p<0.01, * p<0.05. Representative images of MH agar plates showing colony forming units (CFU) for E. coli, P. aeruginosa and S. aureus before (top) and after (bottom) treatment with cPR2 at 2x MIC after 3 h (B). CLSM images of Live/Dead bacteria before (top) and after (bottom) cPR2 treatment for 2 h. SYTO 13 (green) was used to label both live and dead bacteria and propidium iodide (red) was the stain used to identify dead bacteria (C); scale bar: 10 μm.

Fig. 4

3D-SIM images of E. coli ATCC 25922 bacteria before (A) and after treatment with 200 μg/mL FITC-cPR2 in MHB for 90 min (B); scale bar: 2 μm. SEM of E. coli ATCC 25922 bacteria before (C) and after treatment with cPR2 for 90 min with arrows pointing to large holes in the bacterial membrane (D); scale bar: 2 μm. Nitrocefin assay was utilized to monitor OM permeability in the presence (200 μg/mL) and absence of cPR2 (E); IM permeability was evaluated by measuring β-galactosidase activity in the presence (200 μg/mL) and absence of cPR2 (F).

Scheme 1

Graphical illustration for preparing cationic polyrotaxanes (cPRs) with variable charge densities. All the hydroxyl groups of a-CD on PR were successfully activated with CDI for efficient conjugation to Boc-EDA as the positive ligand (R = a) and ethanolamine as the neutral ligand (R = b). At the end of the conjugation reaction, Boc protecting groups were removed with concentrated aqueous HCl to yield the final four cPRs possessing varying surface charge properties: cPR0, cPR1, cPR2, and cPR3