Healthcare-associated infections are often challenging to treat as bacteria can rapidly adapt to conventional antibiotics. Therefore, alternative efficient antibacterials are needed. We hypothesized that combining nanosilver with chitosan would yield synergistically acting nanocomposites that could be used in topical treatments (e.g., wound dressings) and surface coatings, whereas the higher the share of chitosan, the higher the synergistic potency. To prove that, three silver–chitosan nanocomposites (nAgCSs) with different Ag-to-chitosan weight ratios (1:0.3, 1:1, and 1:3) were synthesized, physicochemically characterized and evaluated for their antibacterial potency toward clinically critical Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and Gram-positive bacteria Staphylococcus aureus. Experiments were conducted in deionized water to minimize the speciation effects of silver. The nAgCSs proved highly antibacterial against E. coli and P. aeruginosa, whereas the nAgCSs with the highest chitosan share were the most potent (24-h MBC values 0.07–0.28 mg Ag/L). As chitosan is a polycationic polymer, we assumed the synergy observed was mainly driven by chitosan attaching to the negatively charged bacterial cells and provoking increased local shedding of silver ions. Our hypothesis was proven by analyzing the contacts between the nAgCSs and bacteria using confocal laser scanning microscopy and flow cytometry and quantifying the bioavailability of silver from the nAgCSs by an Ag-sensing biosensor. To our knowledge, this type of combined hypothesis-driven study on silver–chitosan nanocomposites’ synergistic properties has not been conducted before.

Copper and chitosan are used for biomedical applications due to their antimicrobial properties. In this study, a facile method for the synthesis of chitosan-copper oxide nanocomposites (nCuO-CSs) was modified, yielding stable colloidal nCuO-CSs suspensions. Using this method, nCuO-CSs with different copper-to-chitosan (50–190 kDa) weight ratios (1:0.3, 1:1, 1:3) were synthesized, their physicochemical properties characterized, and antibacterial efficacy assessed against Gram-negative Escherichia coli and Pseudomonas aeruginosa, and Gram-positive Staphylococcus aureus. The nCuO-CSs with a primary size of ∼10 nm and a ζ-potential of >+40 mV proved efficient antibacterials, acting at concentrations around 1 mg Cu/L. Notably, against Gram-negative bacteria, this inhibitory effect was already evident after a 1-h exposure and surpassed that of copper ions, implying to a synergistic effect of chitosan and nano-CuO. Indeed, using flow cytometry and confocal laser scanning microscopy, we showed that chitosan promoted interaction between the nCuO-CSs and bacterial cells, facilitating the shedding of copper ions in the close vicinity of the cell surface. The synergy between copper and chitosan makes these nanomaterials promising for biomedical applications (e.g., wound dressings).

All novel materials should be analyzed for their potential environmental hazard. In this study, the toxicity of different silver–chitosan nanocomposites—potential candidates for wound dressings or antimicrobial surface coatings—was evaluated using environmentally relevant aquatic microcrustaceans Daphnia magna and Thamnocephalus platyurus and naturally luminescent bacteria Vibrio fischeri. Three silver-chitosan nanocomposites (nAgCSs) with different weight ratios of Ag to CS were studied. Citrate-coated silver nanoparticles (nAg-Cit), AgNO₃ (ionic control) and low molecular weight chitosan (LMW CS) were evaluated in parallel. The primary size of nAgCSs was ~50 nm. The average hydrodynamic sizes in deionized water were ≤100 nm, and the zeta potential values were positive (16–26 mV). The nAgCSs proved very toxic to aquatic crustaceans: the 48-h EC₅₀ value for D. magna was 0.065–0.232 mg/L, and the 24-h LC₅₀ value for T. platyurus was 0.25–1.04 mg/L. The toxic effect correlated with the shedding of Ag ions (about 1%) from nAgCSs. Upon exposure of V. fischeri to nAgCSs for 30 min, bacterial luminescence was inhibited by 50% at 13–33 mg/L. However, the inhibitory effect (minimum bactericidal concentration, MBC) on bacterial growth upon 1 h exposure was observed at higher concentrations of nAgCSs, 40–65 mg/L. LMW CS inhibited bacterial luminescence upon 30-min exposure at 5.6 mg/L, but bacterial growth was inhibited at a much higher concentration (1 h MBC > 100 mg/L). The multi-trophic test battery, where D. magna was the most sensitive test organism, ranked the silver-chitosan nanocomposites from ‘extremely toxic’ [L(E)C₅₀ ≤ 0.1 mg/L] to ‘very toxic’ [L(E)C₅₀ > 0.1–1 mg/L]. Chitosan was toxic (EC(L)₅₀) to crustaceans at ~12 mg/L, and ranked accordingly as ‘harmful’ [L(E)C₅₀ > 10–100 mg/L]. Thus, silver-chitosan nanocomposites may pose a hazard to aquatic organisms and must be handled accordingly.