Synthesis and Antibiotic Activity of Chitosan-Based Comb-like Co-Polypeptides

Abstract

Infections caused by multidrug-resistant Gram-negative bacteria have been named one of the most urgent global health threats due to antimicrobial resistance. Considerable efforts have been made to develop new antibiotic drugs and investigate the mechanism of resistance. Recently, Anti-Microbial Peptides (AMPs) have served as a paradigm in the design of novel drugs that are active against multidrug-resistant organisms. AMPs are rapid-acting, potent, possess an unusually broad spectrum of activity, and have shown efficacy as topical agents. Unlike traditional therapeutics that interfere with essential bacterial enzymes, AMPs interact with microbial membranes through electrostatic interactions and physically damage cell integrity. However, naturally occurring AMPs have limited selectivity and modest efficacy. Therefore, recent efforts have focused on the development of synthetic AMP analogs with optimal pharmacodynamics and an ideal selectivity profile. Hence, this work explores the development of novel antimicrobial agents which mimic the structure of graft copolymers and mirror the mode of action of AMPs. A family of polymers comprised of chitosan backbone and AMP side chains were synthesized via the ring-opening polymerization of the N-carboxyanhydride of l-lysine and l-leucine. The polymerization was initiated from the functional groups of chitosan. The derivatives with random- and block-copolymer side chains were explored as drug targets. These graft copolymer systems exhibited activity against clinically significant pathogens and disrupted biofilm formation. Our studies highlight the potential of chitosan-graft-polypeptide structures in biomedical applications.

Keywords: N-carboxyanhydrides; antimicrobial peptides; chitosan; comb-like co-polypeptide; ring-opening polymerization.

Figure 1

Synthetic methodology developed to fabricate cationic peptidopolysaccharide graft copolymers consisting of L-lysine and L-leucine oligomers conjugated to chitosan via ring-opening polymerization of N-carboxyanhydrides (NCA-ROP).

Figure 2

¹H-NMR spectrum of the CHI-CSA in DMSO-d₆ with schematic presentation of repeat unit.

Figure 3

NCA-ROP synthesis of CHI-based GCPs.

Figure 4

Tentative mechanism of the NCA-ROP using CHI-CSA as macroinitiator.

Figure 5

¹H NMR spectra (DMSO-d₆) of CHI-graft-poly(l-lysine(Z)).

Figure 6

¹H NMR spectra (DMSO-d₆) of CHI-graft-poly (l-leucine-co-l-lysine).

Figure 7

Synthesis of CHI-graft-poly(L-leucine-block-L-lysine) (“Block”) via sequential ring-opening polymerization of L-leucine (a) and L-lysine(Z) (b) in a two-step process.

Figure 8

¹H NMR spectra (DMSO-d₆) of CHI-graft-poly(l-leucine-block-l-lysine).

Figure 9

Representative FTIR spectra of CHI-CSA (blue), CHI-graft-poly(L-lysine(Z)) (red), and CHI-graft-poly(l-lysine) (black).

Figure 10

SEC of CHI-graft-poly(L-lysine) (black) and a linear model compound GlcN-term-poly(L-lysine) (red) in DMF.

Figure 11

Growth inhibition curve of E. coli treated with different concentrations of GlcN-term-poly(l-lysine) (black squares), CHI-graft-poly(l-lysine) (red circles), CHI-graft-poly(l-leucine-co-l-lysine) (blue triangles), and CHI-graft-poly(l-leucine-block-l-lysine) (green triangles) for 24 h. Data are presented as the mean and standard deviation, n = 12. The half maximal inhibitory concentrations, IC₅₀, of tested GCPs estimated from these data are as follows: GlcN-term-poly(l-lysine—0.10 mg/mL, CHI-graft-poly(l-leucine-block-l-lysine)—0.51 mg/mL, CHI-graft-poly(l-leucine-co-l-lysine)—0.66 mg/mL, and CHI-graft-poly(l-lysine)—0.85 mg/mL.

Figure 12

Growth inhibition curve of S. aureus treated with different concentrations of GlcN-term-poly(l-lysine) (black squares), CHI-graft-poly(l-lysine) (red circles), CHI-graft-poly(l-leucine-co-l-lysine) (blue triangles), and CHI-graft-poly(l-leucine-block-l-lysine) (green triangles) for 24 h. Data are presented as the mean and standard deviation, n = 12.

Figure 13

Absorbance of the graft copolymers at varying concentrations in MH media at 600 nm: GlcN-term-poly(l-lysine) (black squares), CHI-graft-poly(l-lysine) (red circles), CHI-graft-poly(l-leucine-co-l-lysine) (blue triangles), and CHI-graft-poly(l-leucine-block-l-lysine) (green triangles) for 24h. Data are presented as the mean ± standard deviation, n = 4.

Figure 14

Inhibition of adherent biomass upon exposure to CHI-based GCPs. A. tumefaciens was grown in the presence of the respective graft copolymers at a concentration of 0.25 mg/mL. The ΔvisR and Δupp mutants exhibited enhanced and depleted film formation, respectively. Total adherent biomass was quantified by crystal violet staining. Mean values of three independent experiments and standard error are shown.

Figure 15

Optical density of A. tumefaciens planktonic biomass treated with 0.25 mg/mL of each polymer. Values are averages of triplicate assays and error bars represent standard deviation.

Figure 16

Changes in adherent biofilm mass (A₆₀₀) of A. tumefaciens normalized by planktonic biomass (OD₆₀₀) treated with 0.25 mg/mL of each polymer. Values are averages of triplicate assays and error bars represent standard deviation. Data for each strain are normalized (100%) to non-treated cultures of each strain (black bars).