Polyphenol-functionalized Fe 3 O 4 nanomaterials for combating multidrug-resistant ESKAPE pathogens

1 Introduction

Antimicrobial resistance (AMR) is unequivocally recognized as one of the defining biomedical threats of the twenty-first century. The landmark 2019 Global Burden of Disease systematic analysis estimated that bacterial AMR was directly responsible for 1.27 million deaths (95% UI: 0.911–1.71 million) and associated with 4.95 million deaths worldwide (Murray et al., 2022; GBD 2021 Antimicrobial Resistance Collaborators, 2024). Crucially, lower respiratory infections—frequently caused by Gram-negative pathogens in critical care settings—accounted for over 1.5 million associated deaths and approximately 50 million disability-adjusted life years (DALYs). Among the six leading pathogens responsible for 929,000 attributable deaths, three are central to the present investigation: Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Subsequent forecasting models project that annual mortality directly attributable to AMR will reach 1.91 million by 2050, with a cumulative toll exceeding 39 million attributable deaths between 2025 and 2050 (Naghavi et al., 2024). In parallel, the World Health Organization continues to classify AMR among the most urgent global health priorities, emphasizing that therapeutic erosion compromises not only infectious disease care but also surgery, oncology, intensive care, and transplantation medicine (World Health Organization, 2024).

Within this broader AMR landscape, ESKAPE pathogens are particularly alarming because they combine extensive intrinsic defenses with a remarkable capacity to acquire new resistance determinants. Prior research has underscored the diverse mechanisms of resistance and their increasing clinical significance (De Oliveira et al., 2020; Miller and Arias, 2024). Although the classical ESKAPE acronym includes both Gram-positive and Gram-negative species, the present manuscript focuses on K. pneumoniae, A. baumannii, and P. aeruginosa, because these opportunistic Gram-negative pathogens are major drivers of healthcare-associated pneumonia, device-related infection, bloodstream infection, chronic wound infection, and ventilator-associated disease in settings where multidrug-resistance and extensively drug-resistance severely restrict therapeutic choice (De Oliveira et al., 2020; Miller and Arias, 2024). The clinical challenge posed by these organisms cannot be explained solely by conventional resistance genes. Biofilm-associated growth is a major virulence trait in ESKAPE infections, because the biofilm matrix creates diffusion barriers, metabolic heterogeneity, and spatial protection that reduce antibiotic effectiveness and favor chronicity (Tiwari, 2023). Simultaneously, persister cells contribute to the recalcitrance of long-lasting bacterial infections by surviving otherwise lethal antibiotic exposure without necessarily carrying stable genetic resistance (Fisher et al., 2017). Consequently, effective anti-infective systems should ideally operate across several biological levels at once, including membrane interaction, metabolic tolerance, intercellular communication, and structured community formation.

Natural polyphenols are promising candidates for this purpose because their chemical diversity supports pleiotropic antimicrobial behavior. Previous studies have shown that structurally distinct phenolic compounds may converge on related biological processes involved in biofilm suppression (Buchmann et al., 2023). Curcumin specifically, has shown robust antimicrobial potential while also emphasizing strain dependence, physicochemical instability, and translational limitations linked to poor aqueous dispersibility and limited bioavailability (Adamczak et al., 2020; Hussain et al., 2022). Quercetin has been reported to interfere with multiple bacterial targets, including membrane-related processes, oxidative balance, and intracellular signaling (Nguyen and Bhattacharya, 2022). Gallic acid stands out particularly because it can directly interfere with quorum sensing and biofilm-associated behavior, reinforcing its relevance as an anti-virulence scaffold (Dai et al., 2026).

Despite this, the practical deployment of free polyphenols remains limited by instability in aqueous environments, rapid degradation, restricted site-specific delivery, and suboptimal persistence at infected interfaces. Previous research has demonstrated that nanoformulation strategies can substantially improve the antibacterial performance of polyphenols by improving cargo protection, interfacial reactivity, and controllable biological interaction (Guo et al., 2021; Liu et al., 2023). From a nanomaterials perspective, this means that formulation is not merely a vehicle issue; it directly reshapes the structure–property–function relationship of the bioactive system.

Among candidate inorganic platforms, magnetite is especially relevant because Fe₃O₄ nanoparticles combine magnetic responsiveness, surface tunability, colloidal adaptability, and broad biomedical compatibility. Fe₃O₄ systems are widely considered significant for biomedical applications, due to their favorable magnetic properties and amenability to surface functionalization (Ganapathe et al., 2020). While recent advances in nanomaterial engineering have enabled the synthesis of magnetite in diverse anisotropic morphologies—including cubic architectures that exhibit enhanced saturation magnetization due to reduced surface spin disorder (Wang et al., 2020), rod-shaped particles with unique directional magnetic behavior (Marcuello et al., 2018), and multi-core nanoflower assemblies optimized for hyperthermia applications (Theodosiou et al., 2022) — the present study deliberately selected a spherical (globular) geometry. This morphological selection is predicated on three fundamental physicochemical considerations: (i) the isotropic surface curvature ensures a uniform and reproducible ligand grafting density, which is critical for consistent biological performance across a panel of clinical isolates; (ii) spherical nanoparticles synthesized via alkaline co-precipitation exhibit favorable colloidal stability in aqueous biological media, as confirmed by the narrow hydrodynamic diameter distribution (42–45 nm) and highly negative zeta potential (−28 to −35 mV) reported herein; and (iii) the co-precipitation route affords a highly scalable, single-step synthesis without the requirement for high-temperature decomposition solvents typically necessary for anisotropic morphologies. In parallel, phenolic compounds present a versatile route for nanoparticle engineering by promoting colloidal stability, interfacial control, and biologically responsive performance (Cardoso et al., 2017). From a materials-engineering perspective, phenolic-enabled nanotechnology is particularly relevant, as phenolic chemistry enables versatile nanoparticle design and interfacial stabilization in biomedical applications (Wu et al., 2021). Moreover, polyphenol-functionalized nanoparticles have been observed to exhibit improved antimicrobial and antibiofilm properties compared to their unconjugated counterparts (Velázquez-Lam et al., 2020).

Collectively, the literature suggests that coupling polyphenols with Fe₃O₄ can generate multifunctional nanomaterials able to overcome several limitations of free phytochemicals while exploiting the anti-biofilm and anti-virulence potential of phenolic chemistry. On this basis, the present study aimed to synthesize Fe₃O₄@Polyphenol nanomaterials functionalized with gallic acid, curcumin, or quercetin; to characterize their principal physicochemical properties; and to evaluate how these properties relate to antimicrobial, anti-biofilm, anti-persister, quorum-quenching, and efflux-pump-modulating effects against clinical multidrug-resistant/extensively drug-resistant isolates of K. pneumoniae, A. baumannii, and P. aeruginosa.

From a translational perspective, given the robust antibiofilm and anti-persister efficacy required to eradicate recalcitrant Gram-negative infections at localized sites, the primary intended clinical application of these nanomaterials is the treatment of complicated skin and soft tissue infections (cSSTIs) and chronic wound infections, including diabetic foot ulcers and burn wounds colonized by multidrug-resistant pathogens. A secondary application domain encompasses device-associated biofilm infections, particularly ventilator-associated pneumonia and catheter-related bloodstream infections, where the magnetic properties of the Fe₃O₄ core could facilitate targeted delivery or surface immobilization.

2 Materials and methods

2.1 Synthesis of Fe₃O₄-polyphenol nanomaterials

The nanomaterials were synthesized by an optimized in situ alkaline co-precipitation method (Figure 1). Ferric and ferrous salts were dissolved in deionized water at a 2:1 M ratio under nitrogen atmosphere to limit uncontrolled oxidation during magnetite formation. Gallic acid, curcumin, or quercetin were then introduced into the precursor system so that organic functionalization occurred concomitantly with nanoparticle generation. Precipitation was induced by the gradual addition of ammonium hydroxide under vigorous magnetic stirring at ambient temperature. The resulting black precipitate was magnetically recovered, washed repeatedly with deionized water and ethanol to remove residual precursors and non-adsorbed material, and subsequently lyophilized to improve storage stability.

2.2 Physicochemical characterization

The crystalline structure was examined by X-ray diffraction (XRD) using Cu Kα radiation (λ = 1.5406 Å) over a 2θ range of 20°–70° with a step size of 0.02° and a scan rate of 2°/min; average crystallite dimensions were estimated via the Scherrer equation. Fourier transform infrared spectroscopy (FTIR) was performed in attenuated total reflectance (ATR) mode over a spectral range of 4,000–400 cm^-1 with a resolution of 4 cm^-1 and 64 co-added scans to confirm the presence of Fe–O bonds and identify characteristic bands associated with each polyphenolic coating. Transmission electron microscopy (TEM) was conducted at an accelerating voltage of 200 kV in bright-field imaging mode; particle size distribution was determined from measurements of ≥200 individual particles using ImageJ software. Dynamic light scattering (DLS) and zeta potential measurements were performed at 25 °C with a measurement angle of 173° (backscatter) and an equilibration time of 120 s; disposable folded capillary cells were used for zeta potential determination. Thermogravimetric analysis (TGA) was performed under a nitrogen atmosphere (flow rate: 50 mL/min) with a heating rate of 10 °C/min from 25 °C to 800 °C to quantify the mass fraction of organic material associated with the Fe₃O₄ core. Image acquisition and assembly were performed using the instrumentation software associated with the characterization platforms, and figure preparation was limited to uniform layout adjustments applied without selective manipulation.

2.3 Antimicrobial and antibiofilm evaluation

A total of 32 clinical multidrug-resistant/extensively drug-resistant isolates were evaluated, including 12 K. pneumoniae, 10 A. baumannii, and 10 P. aeruginosa strains recovered from severe nosocomial infections. Antimicrobial susceptibility testing was performed by broth microdilution in accordance with Clinical and Laboratory Standards Institute guidance. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were determined for each polyphenol-coated Fe₃O₄ formulation, for the corresponding free polyphenols, and for bare Fe₃O₄ nanoparticles used as comparators.

The inhibitory effect on biofilm formation was assessed at sub-inhibitory concentrations using the crystal violet assay for biomass quantification. Mature 48 h biofilms were additionally evaluated by crystal violet staining and by XTT reduction to determine metabolic viability after exposure to the nanomaterials. To assess anti-persister activity, antibiotic-tolerant subpopulations were treated with the tested formulations, and the remaining viable burden was quantified as log₁₀ colony-forming units per milliliter.

Persister cells were generated using an aminoglycoside challenge protocol adapted from established methodologies (Grassi et al., 2017; Soares et al., 2019). Briefly, overnight cultures of each clinical isolate were diluted 1:100 in fresh cation-adjusted Mueller-Hinton broth (CAMHB) and grown to stationary phase (18h, 37 °C, 200 rpm), yielding an initial bacterial density of approximately 10⁹ CFU/mL. These stationary-phase cultures were subsequently challenged with gentamicin at 100× the strain-specific MIC for 4 h at 37 °C under continuous agitation (200 rpm), effectively eliminating the metabolically active susceptible majority and enriching the antibiotic-tolerant persister subpopulation. Following the antibiotic challenge, surviving cells were harvested by centrifugation (5,000×g, 10 min), washed twice with sterile phosphate-buffered saline (PBS, pH 7.4) to remove residual gentamicin, and resuspended in fresh CAMHB. The persister-enriched suspension was then exposed to the Fe₃O₄@polyphenol nanomaterials at 4× MIC for an additional 4 h at 37 °C. Viable counts were determined by serial ten-fold dilution in PBS and plating on Mueller-Hinton agar (incubation: 24h, 37 °C). Persister cell reduction was expressed as Δlog₁₀ CFU/mL relative to untreated persister controls. All experiments were performed in biological triplicate with technical duplicates.

2.4 Gene expression analysis

The transcriptional effects of the nanomaterials on key virulence-associated pathways were examined by quantitative reverse transcription-polymerase chain reaction. The targeted genes included quorum-sensing regulators such as lasI, lasR, rhlI, and rhlR, as well as major efflux-pump components including mexA, mexB, adeA, and adeB. Relative expression values were normalized to housekeeping reference genes and interpreted as fold-change in comparison with untreated controls.

2.5 Cytocompatibility analysis

In vitro cytocompatibility was evaluated by MTT assay using human dermal fibroblasts and Caco-2 epithelial cells after 24h and 48 h exposure to increasing concentrations of the nanomaterials. The purpose of this analysis was to determine whether antibacterial concentrations overlapped with concentration ranges maintaining acceptable mammalian-cell viability.

2.6 Statistical analysis

All quantitative data were expressed as mean ± standard deviation derived from at least three independent biological replicates. Statistical significance was determined by a one-way analysis of variance followed by Tukey’s post hoc test. A value of p < 0.05 was considered statistically significant.

3 Results

3.1 Physicochemical characterization of Fe₃O₄-polyphenol nanomaterials

The combined physicochemical analyses consistently supported the successful synthesis of polyphenol-coated Fe₃O₄ nanomaterials XRD patterns confirmed the inverse spinel structure characteristic of magnetite, while the calculated crystallite size remained in the ultra-fine range, at approximately 12 nm. TEM observations showed predominantly spherical particles with a relatively narrow size distribution, supporting the homogeneity of the precipitation process. While scanning electron microscopy (SEM) was not performed in the present study due to the unavailability of field-emission SEM instrumentation required for individual nanoparticle imaging in the 12 nm size regime, the convergent evidence from TEM (200 kV, bright-field mode), XRD crystallite analysis, and DLS hydrodynamic measurements collectively provides comprehensive morphological characterization at the relevant nanoscale length scales (Table 1). This multi-technique approach is consistent with current best practices in nanoparticle characterization (Mourdikoudis et al., 2018; Modena et al., 2019). FTIR spectra demonstrated the coexistence of Fe–O vibration bands and polyphenol-associated signals, confirming surface functionalization rather than simple physical mixing. DLS measurements indicated average hydrodynamic diameters between 42 and 45 nm, whereas the zeta potential remained strongly negative (−28 to −35 mV), consistent with good colloidal stability. TGA further indicated an organic loading of approximately 15%–20%. The main physicochemical parameters are summarized in Table 1.

TABLE 1. Physicochemical properties of Fe₃O₄ polyphenol nanobiocomposites.

These results indicate that polyphenolic functionalization did not compromise magnetite phase formation while introducing an organic interfacial layer capable of modulating dispersion behavior and biological performance. In nanomaterials terms, the generated systems retained the core structural signature of Fe₃O₄ while acquiring a surface chemistry likely to influence colloidal persistence, target interaction, and interfacial availability in aqueous biological environments. Importantly, during the in situ functionalization process, no macroscopic aggregation or irreversible flocculation was observed. The polydispersity index (PDI) for all three functionalized formulations remained below 0.25, and the suspensions were stable for over 72 h without visible sedimentation. In contrast, bare Fe₃O₄ nanoparticles exhibited rapid sedimentation and a PDI exceeding 0.4 in the absence of the polyphenol coating. The robust colloidal stability of the functionalized nanomaterials is attributed to the electrosteric repulsion conferred by the polyphenol corona: the highly negative zeta potential (−28 to −35 mV) provides electrostatic repulsion, while the organic layer introduces a steric barrier that prevents direct surface-to-surface contact between adjacent magnetite cores.

3.2 Antibacterial activity against MDR/XDR isolates

All tested Fe₃O₄@Polyphenol nanomaterials displayed stronger antimicrobial activity than their corresponding free polyphenols. Among them, Fe₃O₄@gallic acid exhibited the highest potency, with MIC values ranging from 16 to 64 μg/mL across the tested strains. Relative to free gallic acid, this represented a 4- to 8-fold improvement in inhibitory activity. Acinetobacter baumannii emerged as the most susceptible species in the studied panel, whereas P. aeruginosa retained the highest intrinsic resistance, which is consistent with its known permeability barriers and active efflux capacity (Supplementary Figure S1 confirms the Gram-negative coccobacillary morphology of the tested isolates). Comparative MIC and MBC results are presented in Table 2, and the interspecies distribution of MIC values is illustrated in Figure 2.

TABLE 2. Comparative MIC and MBC Values (µg/mL) of Fe₃O₄@Polyphenol Nanobiocomposites vs. Free Polyphenols and Bare Fe₃O₄ Against MDR/XDR ESKAPE Pathogens.

The superiority of the nanoformulations was also evident for curcumin, whose poor aqueous solubility often restricts measurable antimicrobial performance in free form. The enhanced performance of curcumin-coated Fe₃O₄ nanoparticles strongly suggests that nanoengineering improved the effective biological presentation of the phytochemical cargo.

3.3 Inhibition of biofilm formation and reduction of mature biofilms

At MIC/2 concentrations, all tested nanomaterials significantly inhibited early biofilm formation. Curcumin-coated Fe₃O₄ nanoparticles showed the strongest activity in this assay, inhibiting biofilm biomass by 82.3% in K. pneumoniae, 88.7% in A. baumannii, and 76.8% in P. aeruginosa (Figure 3). Since these effects were observed under sub-inhibitory conditions, these likely indicate interference with biofilm-associated processes rather than merely growth suppression (representative microscopy images documenting biofilm inhibition are provided in Supplementary Figures S1A-B).

The formulations also maintained effectiveness against established 48 h biofilms. Mature-biofilm biomass was reduced by up to 78%, while metabolic viability decreased by up to 85% (Figure 4), demonstrating efficacy against highly tolerant sessile communities (see Supplementary Figures S1C-E for visual documentation of biofilm disruption and nanoparticle–biofilm interaction).

3.4 Reduction of persister-cell burden

In the persister-cell model, Fe₃O₄@gallic acid reduced the viable tolerant subpopulation by 2.5–5.0 log₁₀ CFU/mL (Figure 5). These findings suggest effectiveness not only against actively dividing cells but also against phenotypic variants that are frequently associated with treatment failure and relapse.

3.5 Modulation of quorum-sensing and efflux-associated pathways

Mechanistic analysis by qRT-PCR showed that the phenotypic anti-biofilm and anti-persister effects were accompanied by transcriptional suppression of key regulatory pathways. In P. aeruginosa, the treatment with nanomaterials induced a significant downregulation of the quorum-sensing regulators lasR and rhlR, with fold reductions ranging from 2.5 to 6.8 (p < 0.01). The expression of the major efflux-associated gene mexB was notably reduced. In A. baumannii, the efflux component associated with resistance, adeB was also significantly downregulated following treatment (Figure 6). A consolidated overview of anti-biofilm, anti-persister, and transcriptional outcomes is provided in Table 3.

TABLE 3. Summary of Anti-Biofilm and Anti-Persister Activity of Fe₃O₄@Polyphenol Nanobiocomposites Against ESKAPE Pathogen maximum/minimum values explicitly reported in the text.

MDR = Multidrug Resistant; XDR = Extensively Drug-Resistant; QS = Quorum Sensing; CFU = Colony-Forming Units.

3.6 In vitro cytocompatibility and therapeutic window

The transcriptional findings are consistent with a multi-target mechanism of action for the polyphenol-coated Fe₃O₄ systems, indicating effects beyond direct growth inhibition toward attenuation of communication networks and transport pathways associated with Gram-negative adaptive resilience.

In MTT assays conducted on human dermal fibroblasts, cell viability remained above 80% even at 256 μg/mL, a concentration exceeding the MIC values observed for many of the tested isolates (Figure 7). This separation between antibacterial activity and overt mammalian-cell toxicity suggests a promising therapeutic window, particularly for local or surface-associated applications where high local concentrations may be desirable.

Although in vitro cytocompatibility does not substitute for comprehensive toxicological assessment, these data provide an important preliminary indication that the antimicrobial effects were not achieved only at concentration levels incompatible with mammalian-cell survival.

4 Discussion

The presented findings support the concept that polyphenol-functionalized Fe₃O₄ nanoparticles can act as a hybrid anti-infective platform in which interfacial design directly enhances antibacterial performance. The increased activity concerning free polyphenols is unlikely due to a singular mechanism. Instead, it likely results from the synergistic effects of improved dispersibility, increased local concentration at bacterial surfaces, prolonged functional persistence, and concurrent disruption of multiple bacterial stress-response pathways. These observations align with earlier studies indicating that nanoparticle systems can potentiate antimicrobial activity through enhanced cell-envelope interactions and physicochemical stress induction (Wang et al., 2017).

The structural characteristics of the synthesized materials offer a plausible foundation for their biological performance. The retention of the magnetite phase, nanoscale particle size, negative zeta potential, and measurable organic loading together indicate the formation of a stable organic-inorganic interface instead of merely a basic mixture of components. Such features may enhance colloidal persistence, diffusion through extracellular polymeric matrices, and productive contact with bacterial envelopes, all of which are significant for antibiofilm activity (Shkodenko et al., 2020). Furthermore, the spectroscopic data provide critical insights into the interfacial binding mechanism governing the polyphenol–magnetite interaction. The significant attenuation and red-shifting of the broad O–H stretching band (∼3,300 cm^-1), coupled with the perturbation of the Fe–O vibration at ∼580 cm^-1 in the FTIR spectra, strongly suggest that the polyphenols are not merely physisorbed via weak electrostatic forces. Instead, these spectral shifts are diagnostic of coordinative (chemisorptive) bonding, wherein the ortho-dihydroxyl (catechol) or trihydroxyl (gallol) moieties of the polyphenols form bidentate or bridging coordination complexes with surface Fe³⁺ ions. This coordinative anchoring confers high stability to the organic corona under physiological conditions and is consistent with the robust colloidal behavior observed over extended storage periods. We acknowledge, however, that definitive deconvolution of the Fe 2p and O 1s binding states would require X-ray photoelectron spectroscopy (XPS), which represents a limitation of the current study and a priority for future high-resolution interfacial characterization.

The strong inhibition of early biofilm development, especially for Fe₃O₄@curcumin, further supports the importance of nanostructural optimization. The systems evaluated not only diminished biofilm biomass but also suppressed regulatory pathways linked to organized community behaviour. This comprehensive response provides more insight than biomass reduction alone, indicating a functional disruption of biofilm physiology rather than just superficial structural harm (Sarkar et al., 2024).

The downregulation of lasR and rhlR provides molecular support for the observed impairment of biofilm formation observed in P. aeruginosa, where quorum sensing governs maturation, matrix production, and virulence expression (Hu et al., 2024). Simultaneously, the suppression of mexB and adeB indicates that the nanoformulations might also attenuate adaptive resistance pathways. Reduced efflux capacity could enhance the effectiveness of the nanoformulations by increasing intracellular retention of active phytochemical species (Gupta et al., 2017).

The comparative behavior of gallic acid, curcumin, and quercetin following nanoformulation is especially noteworthy. The available literature suggests that these polyphenols differ in their physicochemical properties and primary antimicrobial mechanisms. Gallic acid is linked to interference with quorum-sensing and anti-virulence effects, while curcumin is recognized for its potential antibacterial properties but also for important translational limitations including low aqueous solubility, instability and restricted bioavailability (Adamczak et al., 2020; Hussain et al., 2022; Dai et al., 2026). Instead of identifying a single dominant compound, the current data suggest that specialization is dependent on the formulation. Fe₃O₄@gallic acid showed the strongest efficacy in MIC reduction and persister killing, whereas Fe₃O₄@curcumin exerted the most pronounced inhibition of initial biofilm formation. This distinction may be useful for future application-specific optimization of the nanomaterial platform (Dai et al., 2026).

From a nanomedicine perspective, the present findings align with the emerging view that effective antimicrobial nanoparticles should blend mechanistic versatility with practical applicability (Elbehiry and Abalkhail, 2025). In this context, polyphenol-coated Fe₃O₄ systems are particularly promising in this regard, as they combine a bioactive organic element with a magnetically responsive inorganic core that is widely used in biomedical fields (Cardoso et al., 2017; Ganapathe et al., 2020; Elbehiry and Abalkhail, 2025). This hybrid character creates opportunities for topical formulations, anti-infective coatings, wound dressings, and possibly magnetically assisted local delivery systems.

Biocompatibility remains a key translational filter for antimicrobial nanomaterials. For Fe₃O₄-based systems, nanoparticle toxicity has been shown to depend on particle size and is closely linked to oxidative-stress pathways (Liu et al., 2016). Within this framework, maintaining fibroblast viability above 80% at concentrations exceeding many MIC values is promising. Nevertheless, these results should be considered preliminary and additional assessments are required in more complex biological models, including long-term exposure, inflammatory profiling, hemocompatibility, and organ-specific cellular systems.

Several limitations must also be acknowledged explicitly. First, although the biological activity profile is strong, the manuscript reports in vitro findings only; therefore, no conclusions can yet be drawn regarding pharmacokinetics, tissue distribution, in vivo tolerability, or therapeutic efficacy in animal infection models. Second, while the qRT-PCR data strongly support anti-virulence action, gene-expression downregulation alone does not fully resolve the sequence of molecular events responsible for growth inhibition and biofilm collapse. Third, the present work focuses on three clinically relevant Gram-negative ESKAPE pathogens, but it does not yet address Gram-positive ESKAPE members or polymicrobial biofilms. Fourth, because the formulations were evaluated against a defined panel of clinical strains, broader multicenter strain collections would be useful to confirm robustness across epidemiologically diverse resistance backgrounds.

Even with these limitations, the translational value of the current dataset is substantial. The study shows that nanostructured phenolic functionalization can move beyond simple antibacterial testing and generate coordinated effects on biofilm formation, persister survival, quorum sensing, and efflux regulation. In the context of AMR, where monofunctional antibacterial agents are increasingly undermined by adaptive resilience, such multi-layered activity is of particular importance. Within this framework, the primary translational target for the polyphenol-functionalized Fe₃O₄ nanosystems described herein is the management of complicated skin and soft tissue infections and chronic wound infections—clinical scenarios in which localized delivery, sustained antibiofilm activity, and anti-persister efficacy are paramount. The demonstrated cytocompatibility on human dermal fibroblasts (viability >80% at concentrations exceeding the therapeutic MIC range) further supports the suitability of these materials for topical wound applications. A secondary application domain encompasses device-associated biofilm infections, where surface immobilization of the nanomaterials could exploit the magnetic responsiveness of the Fe₃O₄ core for targeted coating strategies. The results therefore justify continued preclinical development of polyphenol-coated Fe₃O₄ nanomaterials and support their evaluation as localized anti-infective technologies and antimicrobial biomaterial interfaces.

5 Conclusion

Polyphenol-coated Fe₃O₄ nanomaterials displayed a coherent and therapeutically relevant activity profile against clinical multidrug-resistant/extensively drug-resistant K. pneumoniae, A. baumannii, and P. aeruginosa isolates. Functionalization of magnetite nanoparticles with gallic acid, curcumin, or quercetin enhanced antimicrobial efficacy relative to free polyphenols, while also suppressing biofilm formation, reducing mature-biofilm viability, decreasing persister burden, and down-regulating quorum-sensing and efflux-pump genes. The preservation of acceptable in vitro fibroblast viability further supports the feasibility of these systems as anti-infective nanomaterial platforms. Overall, the study identifies Fe₃O₄@Polyphenol materials as promising candidates for future development in topical therapy, wound management, antimicrobial surface engineering, and related biomaterial applications.

Moving forward, several critical action lines emerge from the present findings. Future research should prioritize: (i) in vivo validation of therapeutic efficacy, biodistribution, and pharmacokinetics using murine chronic wound infection models inoculated with the MDR/XDR clinical isolates characterized herein; (ii) high-resolution interfacial characterization utilizing X-ray photoelectron spectroscopy (XPS) to definitively resolve the coordination chemistry at the polyphenol–magnetite interface; (iii) comprehensive hemocompatibility and immunotoxicity profiling to establish a rigorous safety profile for regulatory translation; (iv) integration of these nanomaterials into functional antimicrobial coatings for medical devices, such as urinary catheters and endotracheal tubes, where biofilm-associated infections by K. pneumoniae, A. baumannii, and P. aeruginosa represent critical clinical challenges; and (v) correlative field-emission SEM imaging for comprehensive morphological characterization of both bare and functionalized nanosystems at the mesoscale.

Among the limitations of the present study, we acknowledge the absence of scanning electron microscopy (SEM) characterization, which would have provided complementary information regarding surface topology and mesoscale inter-particle spatial organization, as well as the lack of X-ray photoelectron spectroscopy (XPS) data that would definitively resolve the interfacial binding mode. These analytical priorities are explicitly included in the future research agenda outlined above.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnano.2026.1871019/full#supplementary-material

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Footnotes

Edited by: Cristina Satriano, University of Catania, Italy

Reviewed by: Carlos Marcuello, Instituto de Nanociencia y Materiales de Aragón (INMA), Spain

Feng Lu, Zhejiang University, China