Unlocking the Potential: PEGylation and Molecular Weight Reduction of Ionenes for Enhanced Antifungal Activity and Biocompatibility.

Numerous synthetic polymers, imitating natural antimicrobial peptides, have demonstrated potent antimicrobial activity, positioning them as potential candidates for new antimicrobial drugs. However, the high activity of these molecules often comes at the cost of elevated toxicity against eukaryotic organisms. In this study, a series of cationic ionenes with varying molecular weights to assess the influence of polymer chain length on ionene activity is investigated. To enhance polymer antimicrobial activity and limit toxicity a PEG side chain is introduced into the repeating unit. The resulting molecules consistently exhibited high activity against three model organisms: E. coli, S. aureus and C. albicans. The incorporation of side PEG chain improves antifungal properties and biocompatibility, regardless of molecular weight. The most important finding of this work is that the reduction of polymer molecular mass led to increased antifungal activity and reduced cytotoxicity against HMF and MRC-5 cell lines simultaneously. As a result, the best-performing molecules reported herein displayed minimal inhibitory concentrations (MIC) as low as 2 and 0.0625 µg mL1 for C. albicans and C. tropicalis respectively, demonstrating exceptional selectivity. It is plausible that some of described herein molecules can serve as potential lead candidates for new antifungal drugs.

Membrane lytic activity of antibacterial ionenes, critical role of phosphatidylcholine (PC) and cardiolipin (CL).

Understanding the mechanism by which an antibacterial agent interacts with a model membrane provides vital information for better design of future antibiotics. In this study, we investigated two antibacterial polymers, hydrophilic C0-T-p and hydrophobic C8-T-p ionenes, known for their potent antimicrobial activity and ability to disrupt the integrity of lipid bilayers. Our hypothesize is that the composition of a lipid bilayer alters the mechanism of ionenes action, potentially providing an explanation for the observed differences in their bioactivity and selectivity. Calcein release experiments utilizing a range of liposomes to examine the impact of (i) cardiolipin (CL) to phosphatidylglycerol (PG) ratio, (ii) overall vesicle charge, and (iii) phosphatidylethanolamine (PE) to phosphatidylcholine (PC) ratio on the activity of ionenes were performed. Additionally, polymer-bilayer interactions were also investigated through vesicle fusion assay and the black lipid membrane (BLM) technique The activity of C0-T-p is strongly influenced by the amount of cardiolipin, while the activity of C8-T-p primarily depends on the overall vesicle charge. Consequently, C0-T-p acts through interactions with CL, whereas C8-T-p modifies the bulk properties of the membrane in a less-specific manner. Moreover, the presence of a small amount of PC in the membrane makes the vesicle resistant to permeabilization by tested molecules. Intriguingly, more hydrophilic C0-T-p retains higher membrane activity compared to the hydrophobic C8-T-p. However, both ionenes induce vesicle fusion and increase lipid bilayer ion permeability.

Polytrimethylenimines: Highly Potent Antibacterial Agents with Activity and Toxicity Modulated by the Polymer Molecular Weight.

Cationic polymers have been extensively investigated as a potential replacement for traditional antibiotics. Here, we examined the effect of molecular weight (MW) on the antimicrobial, cytotoxic, and hemolytic activity of linear polytrimethylenimine (L-PTMI). The results indicate that the biological activity of the polymer sharply increases as MW increases. Thanks to a different position of the antibacterial activity and toxicity thresholds, tuning the MW of PTMI allows one to achieve a therapeutic window between antimicrobial activity and toxicity concentrations. L-PTMI presents significantly higher antimicrobial activity against model microorganisms than linear polyethylenimine (L-PEI) when polymers with a similar number of repeating units are compared. For the derivatives of L-PTMI and L-PEI, obtained through N-monomethylation and partial N,N-dimethylation of linear polyamines, the antimicrobial activity and toxicity were both reduced; however, resulting selectivity indices were higher. Selected materials were tested against clinical isolates of pathogens from the ESKAPE group and Mycobacteria, revealing good antibacterial properties of L-PTMI against antibiotic-resistant strains of Gram-positive and Gram-negative bacteria but limited antibacterial properties against Mycobacteria.

Influence of PEG Subunit on the Biological Activity of Ionenes: Synthesis of Novel Polycations, Antimicrobial and Toxicity Studies.

An alarming increase of antibiotic resistance among pathogens creates an urgent need to develop new antimicrobial agents. Many reported polycations show high antimicrobial activity along with low hemolytic activity. Unfortunately, most of those molecules remain highly cytotoxic against various mammalian cells. In this work, a systematic study on the impact of triethylene glycol monomethyl ether side groups (short polyethylene glycol (PEG) analog) on antimicrobial, hemolytic, and cytotoxic properties of novel amphiphilic ionenes is presented. A detailed description of synthesis, leading to well-defined alternating polymers, which differ in structural elements responsible for hydrophilicity (PEG) and hydrophobicity (alkyl chain), is presented. Obtained results show that the PEG moiety and fine-tuned hydrophilic-lipophilic balance of ionenes synergistically lead to low cytotoxic, low hemolytic molecules with high activity against S. aureus, including methicillin-resistant strains (MRSA). Additionally, the results of mechanistic studies on bacterial cells and fluorescently labeled liposomes are also discussed.

Influence of lipid bilayer composition on the activity of antimicrobial quaternary ammonium ionenes, the interplay of intrinsic lipid curvature and polymer hydrophobicity, the role of cardiolipin.

Incorporation of hydrophobic component into amphiphilic polycations structure is frequently accompanied by an increase of antimicrobial activity. There is, however, a group of relatively hydrophilic polycations containing quaternary ammonium moieties along mainchain, ionenes, which also display strong antimicrobial and limited hemolytic properties. In this work, an influence of a hydrophobic side group length on antimicrobial mechanism of action is investigated in a series of novel amphiphilic ionenes. High antimicrobial activity was found by determination of minimum inhibitory concentration (MIC) and minimum bactericidal, and fungicidal concentration (MBC and MFC) in both growth media and a buffer. Biocompatibility was estimated by hemolytic and mammalian cells viability assays. Mechanistic studies were performed using large unilamellar vesicles (LUVs) with different lipid composition, as simplified models of cell membranes. The investigated ionenes are potent and selective antimicrobial molecules displaying a decrease of antimicrobial activity correlated with increase of hydrophobicity. Studies using LUVs revealed that the cardiolipin is an essential component responsible for the lipid bilayer permeabilization by investigated ionens. In contrast to relatively hydrophilic ionenes, more hydrophobic polymers showed an ability to stabilize membranes composed of lipids with negative spontaneous curvature in a certain range of polymer to lipid ratio. The results substantially contribute to the understanding of antimicrobial activity of the investigated class of polymers.