Amphiphilic silver nanoclusters show active nano–bio interaction with compelling antibacterial activity against multidrug-resistant bacteria

Multidrug-resistant bacteria (MDRB) pose escalating clinical and public health challenges due to their ability to evade broad spectra of antibiotics, leading to difficult-to-eradicate infections and substantial economic burden. Novel therapeutics, including diverse nanomaterials (metallic, nonmetallic, metal oxides, and carbonaceous), have shown MDR bactericidal effects through mechanisms such as membrane damage, reactive oxygen species (ROS) generation, enzyme-mimicking (peroxidase-like) activity, ion release (e.g., Ag+ attacking thiol-rich proteins), and biofilm eradication. Silver nanomaterials (AgNMs), especially silver nanoparticles (AgNPs), are among the most effective antimicrobial nanomedicines, yet their efficacy against MDRB can be limited and resistance may re-emerge after repeated treatments. A critical knowledge gap remains regarding how combinations of physicochemical properties—size, core/shell structure, and surface ligands (governing solubility, charge, stability, hydrophilicity/hydrophobicity)—collectively dictate antimicrobial performance. This study investigates whether small-sized amphiphilic silver nanoclusters (AgNCs) with tailored core structures and ligand shells can enhance nano–bio interactions and achieve superior antibacterial activity against MDR Pseudomonas aeruginosa, both in vitro and in vivo.

Prior work has demonstrated antimicrobial actions of multiple nanomaterial classes via membrane disruption, ROS generation, enzyme-mimicking activity, metal ion release, and biofilm eradication. AgNPs are widely studied but show variable efficacy against MDR strains and potential for resistance with repeated exposure. Most previous studies assessed single physicochemical parameters in isolation (size, surface ligands, charge), yielding no consensus on optimal property combinations and lacking predictive tools for design. To address this, the authors compiled >1700 initial publications via Web of Science and PubMed keyword searches, applied inclusion/exclusion criteria to derive 266 datasets from 36 papers, and extracted variables (size, ligands, charge, Gram type, strain, exposure time, resistance, MIC). This landscape motivated a data-driven approach to identify which parameters most strongly influence MIC and to guide AgNM design.

Data mining and modeling: The authors conducted a systematic literature search (keywords: silver nanoparticles/nanosilver/Ag and antibacterial/antimicrobial/bacterial) across Web of Science and PubMed. Inclusion criteria: undoped Ag nanomaterials <100 nm with clearly measured size; antibacterial evaluation via MIC/efficiency; chemically synthesized with defined surface modifications; clearly described bacterial strains/sources. Exclusions included nonsensible homogeneous systems (e.g., textiles, creams, gels, alloys, plastics, clothing) and green syntheses with unclear surface modifications. From 36 papers, 266 datasets were extracted (size, surface ligand, charge, Gram feature, bacterial strain, exposure time, resistance, MIC). A random forest (RF) model (R package randomForest; 200 repetitions) quantified feature importance using the increase in mean squared error (MSE) upon permutation. Size and surface ligands strongly impacted prediction accuracy, while Gram feature contributed less. A classification ring visualization stratified Gram-positive/negative groups and MICs by particle size classes; linear regression assessed size–MIC correlations. Synthesis and characterization: Ag nanoclusters (AgNCs) were synthesized via a two-step route. Step 1 (core formation): AgNO3 (60 mg) in dichloromethane/methanol (ice bath) with 1-adamantanethiol (S-Adm, 15 mg) and tetraphenylphosphonium bromide (12 mg); after 20 min, NaBH4 (1 mL, 45 mg/mL) and triethylamine (50 μL) were added under vigorous stirring; aged 12 h at 4 °C. The aqueous phase was removed, organic phase washed, and dark green hydrophobic crystals crystallized from CH2Cl2 in one week. Step 2 (ligand exchange to amphiphilic shell): mercaptosuccinic acid (MSA, 0.8 mg) added to 1 mL ethanol suspension of organophilic AgNCs (5 mg); stirred 3 h at room temperature; concentrated ammonia (6 μL) added to precipitate AgNCs; solids centrifuged, washed with ethanol, dissolved in deionized water. Conventional AgNPs were prepared for comparison: AgNPs #1 via AgNO3 (60 mg) in 8 mL DMF, NaBH4 (1 mL, 45 mg/mL) and triethylamine (50 μL), aged 6 h at room temperature, then isolated and dispersed in water. AgNPs #2 were synthesized similarly but with mixed ligands (MSA 6.75 mg and S-Adm 7.50 mg). Materials were characterized by absorption spectroscopy and solubility tests (water vs CH2Cl2) to confirm hydrophilic vs amphiphilic properties. Biological materials: MDR strains of Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli were obtained and maintained with institutional ethics approval; corresponding drug susceptibility profiles were available. Enzyme-like activity assay: Peroxidase-like activity was assessed using a TMB assay. AgNPs/AgNCs were diluted to 512.0 μg/mL in deionized water, then mixed with H2O2 (100 μL, 10 M) and TMB (20 μL, 100 mM in DMSO), and brought to 1 mL total volume with water. Absorbance changes were recorded to quantify catalytic activity. Additional assays (not fully detailed in the excerpt) included ROS generation, membrane integrity, ATP synthesis impact, and molecular dynamics simulations to model nano–membrane interactions.

- Small-sized amphiphilic silver nanoclusters (AgNCs) exhibit superior antibacterial activity against multidrug-resistant Pseudomonas aeruginosa compared with conventional Ag nanoparticles (AgNPs). - Random forest analysis of literature-derived datasets identified particle size and surface ligands as the most important determinants of MIC; Gram status contributed less. - Amphiphilic surface ligands on AgNCs promote strong interactions with bacterial membranes and facilitate endocytic uptake; molecular dynamics simulations corroborate enhanced nano–bio association. - The nanocluster core architecture confers strong peroxide-like (enzyme-mimicking) activity, leading to substantial reactive oxygen species (ROS) production that enhances bactericidal potency. - Mechanistically, AgNCs impair bacterial membranes, elevate oxidative stress, and attenuate key cellular processes such as ATP synthesis, collectively leading to enhanced killing. - In vivo, AgNCs effectively treated P. aeruginosa-induced pneumonia in mice, increasing survival, and demonstrated excellent biocompatibility in treated animals. - Spectroscopic and solubility characterizations confirmed distinct hydrophilic (AgNPs) versus amphiphilic (AgNCs) behaviors; TMB assays indicated higher peroxidase-like activity for AgNCs versus AgNPs (as described qualitatively).

The study addresses a central question in antimicrobial nanomedicine: how to rationally design silver-based nanomaterials that overcome MDR bacterial defenses. By integrating data mining with experimental validation, the authors show that optimizing both size and surface ligand amphiphilicity is critical to maximize antibacterial efficacy. Amphiphilic AgNCs more effectively associate with and traverse bacterial membranes, enhancing local delivery of oxidative and ionic stressors. Their nanocluster architecture exhibits elevated peroxidase-like catalytic activity, amplifying ROS generation, which in turn damages membranes, DNA, and metabolic machinery (e.g., ATP synthesis). These mechanisms act in concert, reducing the likelihood of single-pathway resistance and explaining the observed superiority over conventional AgNPs. The in vivo efficacy against MDR P. aeruginosa pneumonia and good biocompatibility underscore translational relevance and suggest that nano–bio interaction design principles can yield potent alternatives to traditional antibiotics for MDR infections.

This work identifies amphiphilic, small-sized silver nanoclusters as highly effective antimicrobials against MDR P. aeruginosa, outperforming conventional AgNPs. Key contributions include: (i) a literature-informed, machine learning-guided identification of size and surface ligands as dominant determinants of MIC; (ii) synthesis of AgNCs with amphiphilic ligands that enhance membrane interactions and uptake; (iii) demonstration of strong peroxidase-like activity and ROS generation intrinsic to the nanocluster structure; and (iv) validation of potent antibacterial activity in vitro and therapeutic efficacy with biocompatibility in a mouse pneumonia model. Future research could explore spectrum broadening across diverse MDR pathogens, optimization of ligand chemistry for targeted delivery and minimized host toxicity, resistance development under repeated exposure, pharmacokinetics and biodistribution, and combinatorial strategies with antibiotics to further enhance efficacy.