Nanoparticles of Short Cationic Peptidopolysaccharide Self-Assembled by Hydrogen Bonding with Antibacterial Effect against Multidrug-Resistant Bacteria

Abstract

Cationic antimicrobial peptides (AMPs) and polymers are active against many multidrug-resistant (MDR) bacteria, but only a limited number of these compounds are in clinical use due to their unselective toxicity. The typical strategy for achieving selective antibacterial efficacy with low mammalian cell toxicity is through balancing the ratio of cationicity to hydrophobicity. Herein, we report a cationic nanoparticle self-assembled from chitosan-graft-oligolysine (CSM5-K5) chains with ultralow molecular weight (1450 Da) that selectively kills bacteria. Further, hydrogen bonding rather than the typical hydrophobic interaction causes the polymer chains to be aggregated together in water into small nanoparticles (with about 37 nm hydrodynamic radius) to concentrate the cationic charge of the lysine. When complexed with bacterial membrane, these cationic nanoparticles synergistically cluster anionic membrane lipids and produce a greater membrane perturbation and antibacterial effect than would be achievable by the same quantity of charge if dispersed in individual copolymer molecules in solution. The small zeta potential (+15 mV) and lack of hydrophobicity of the nanoparticles impedes the insertion of the copolymer into the cell bilayer to improve biocompatibility. In vivo study (using a murine excisional wound model) shows that CSM5-K5 suppresses the growth of methicillin-resistant Staphylococcus aureus (MRSA) bacteria by 4.0 orders of magnitude, an efficacy comparable to that of the last resort MRSA antibiotic vancomycin; it is also noninflammatory with little/no activation of neutrophils (CD11b and Ly6G immune cells). This study demonstrates a promising new class of cationic polymers-short cationic peptidopolysaccharides-that effectively attack MDR bacteria due to the synergistic clustering of, rather than insertion into, bacterial anionic lipids by the concentrated polymers in the resulting hydrogen-bonding-stabilized cationic nanoparticles.

Combined efficacy of a novel antimicrobial cationic peptide polymer with conventional antibiotics to combat multi-drug resistant pathogens

“…Antimicrobial peptides (AMPs) are of interest as alternatives to traditional antibiotics because of their broad spectrum antimicrobial properties and efficacy against MDR bacteria; however, to date they have met limited success due to their often non-selective toxicity (32–36). We previously reported that chitosan based cationic polymers showed antimicrobial activity against a variety of MDR pathogens (12, 20–22). In this study, we demonstrate that CSM5-K5 is synergistically active with traditional antibiotics against three antimicrobial resistant pathogens growing as biofilms in vitro and in vivo .…”

“…CSM5-K5 is a cationic nanoparticle that is self-assembled from chitosan-graft-oligolysine chains with ultralow molecular weight (1450 Da) that selectively kills bacteria with minimal toxicity toward mammalian cells (12). Hydrogen bonding within CSM5-K5 causes the polymer chains to aggregate into small nanoparticles to concentrate the cationic charge of the lysine.…”

“…Hydrogen bonding within CSM5-K5 causes the polymer chains to aggregate into small nanoparticles to concentrate the cationic charge of the lysine. Upon contact with the bacterial membrane, these cationic nanoparticles synergistically cluster anionic membrane lipids and produce a greater membrane perturbation and antibacterial effect than would be achievable by the same quantity of charge if dispersed in individual copolymer molecules in solution (12). We observed partial of full antimicrobial synergy, against both Gram-negative and Gram-positive pathogens, between CSM5-K5 and nearly every antibiotic that we tested.…”

“…CSM5-K5 was prepared as previously described in (12), with the following modifications. 5 g of low molecular weight chitosan (MW 200KDa) was first dispersed in 100 mL of anhydrous DMF and sonicated at 80°C for 1 hour under Argon protection.…”

“…However, many of these derivatives possess high cationicity and rely on hydrophobicity for improved bacterial interaction, which often lead to mammalian hemolysis and toxicity. To overcome these limitations, we have synthesized a low molecular weight copolymer CSM5-K5 hydrochloride salt (where CSM denotes chitosan monomer repeat units and K denotes lysine amino acid repeat units) with a controlled molecular weight of 1450 Da (12). CSM5-K5 displays low hemolysis and toxicity, is antibacterial against a variety of MDR bacteria, and reduces MRSA bacterial burden in a murine wound infection model by four orders of magnitude (12).…”