Synthesis, Characterization and Broad-Spectrum Bactericidal Effects of Ammonium Methyl and Ammonium Ethyl Styrene-Based Nanoparticles

Abstract

Untreatable infections, growing healthcare costs, and increasing human mortality due to the rising resistance of bacteria to most of the available antibiotics are global phenomena that urgently require the discovery of new and effective antimicrobial agents. Cationic macromolecules, acting as membrane disruptors, are widely studied, and several compounds, including two styrene-based copolymers developed by us (P5 and P7), have proved to possess potent broad-spectrum antibacterial effects, regardless of the resistance profiles of the bacteria. Here, we first reported the synthesis and physicochemical characterization of new cationic nanoparticles (NPs) (CP1 and OP2), obtained by polymerizing the monomers 4-ammoniummethylstyrene (4-AMSTY) and 4-ammoniumethylstyrene (4-AESTY) hydrochlorides, whose structures were designed using the cationic monomers of P5 and P7 as template compounds. The antibacterial activity of CP1 and OP2 was assessed against several Gram-positive and Gram-negative multi-drug resistant (MDR) pathogens, observing potent antibacterial effects for both CP1 (MICs = 0.1–0.8 µM) and OP2 (MICs = 0.35–2.8 µM) against most of the tested isolates. Additionally, time-killing studies carried out with CP1 and OP2 on different strains of the most clinically relevant MDR species demonstrated that they kill pathogens rapidly. Due to their interesting physicochemical characteristics, which could enable their mutual formulation as hydrogels, CP1 and OP2 could represent promising ingredients for the development of novel antibacterial dosage forms for topical applications, capable of overcoming severe infections sustained by bacteria resistant to the presently available antibiotics.

A Self-Forming Hydrogel from a Bactericidal Copolymer: Synthesis, Characterization, Biological Evaluations and Perspective Applications

“…As expected, the UV–Vis spectrum of CP1_1.1-Hgel at concentrations 50 µg/mL (MeOH), showed an absorption at λ max = 252 nm, like that of the monomer M1 used to prepare the copolymer CP1, in turn used to obtain the gel ( Figure 2 ) [ 29 ]. In this way it was established that during the formation of both copolymer and gel no interaction occurred that might affect the chromophore characteristics.…”

“…After having prepared the two-components hydrogel CP1OP2-Hgel currently under investigation over its antibacterial activity, we decided to prepare a one-component hydrogel to avoid, in advance, possible antagonistic effects deriving by the simultaneous presence of both CP1 and OP2. Among CP1 and OP2, CP1 was selected because it showed MICs lower than those of OP2 (MICs = 0.1–0.8 µM) against several MDR clinical isolates, including the species mostly found in hospital settings [ 29 ]. In addition, CP1 was preferred to OP2, because, even though not cross-linked, it demonstrated the ability to form the hydrogel upon simple dispersion in water at a concentration lower than those used to prepare CP1OP2-Hgel, without the need of gelling agents or other additives, thus limiting possible skin-incompatibilities in future topical administrations.…”

“…with respect to the copolymer CP1, which is present at 1.1% wt. On the contrary, the spectrum of the dried sample obtained by heating the soaked gel ( Figure 3 b, black line), was very similar to that of copolymer CP1 ( Figure 3 b, red line) [ 29 ], thus establishing that the interactions that occurred during the formation of the gel did not affect the main functional groups of original CP1. Particularly, weak bands at 1497, 1401, 1254, and 1140 cm −1 provided by the aromatic C=C and C-N stretching bands, and intense bands at 1610 cm −1 standing for the C=O stretching of the amide group of DMAA, were detectable in both spectra.…”

“…By carefully selecting monomers and crosslinkers, hydrogels that have desired capabilities, such as preferred hydrophilicity and porosity, can be developed and employed for antibacterial functions. In this scenario, we exploited two bactericidal cationic polymers (CP1 and OP2) [ 29 ], prepared in parallel with a one-component hydrogel made with only CP1, and a two-components hydrogel formulation containing both CP1 and OP2 as active ingredients, in which CP1 acted also as gelling agent. The study on the two-components hydrogel (CP1OP2-Hgel) has been recently published, along with the cytotoxic profile of the two not-formulated ingredients [ 30 ].…”

“…Additionally, the stability of a 3.0% wt/v CP1-gel was evaluated over four months at room temperature by monitoring its inversion properties. Although dose-dependent cytotoxicity experiments were previously carried out on human normal fibroblast with the not-formulated CP1 [ 30 ], and MICs of pristine CP1 have been recently reported against several MDR bacteria [ 29 ], here, dose-dependent cytotoxicity experiments with samples of CP1-based gel obtained by diluting CP1_1.1-Hgel in PBS were carried out in similar conditions to investigate if the formation of the 3D network affected the cytotoxic behavior of CP1. Similarly, new MICs were determined on serial two-fold dilutions of CP1_1.1-Hgel for the same reason.…”