Fe₃O₄–PNIPAAm Nanocomposites: Preparation Method Determines Antibacterial Efficacy and Physicochemical Performance

Abstract

Magnetic poly(N‑isopropylacrylamide) (Fe₃O₄‑PNIPAAm) nanocomposites are promising for biomedical and environmental applications. We compared three synthesis routes—emulsion polymerisation, in‑situ precipitation, and physical addition—to produce Fe₃O₄‑PNIPAAm‑1, –2, and –3, respectively. Scanning electron microscopy, zeta‑potential, thermogravimetric analysis, vibrating sample magnetometry, and dynamic light scattering revealed that Fe₃O₄‑PNIPAAm‑1 and –2 exhibit superior thermal stability, surface charge, and magnetisation, indicating highly stable colloids. All composites, even at low concentrations, induced significant DNA damage in Escherichia coli and Staphylococcus aureus and reduced cell viability. Fe₃O₄‑PNIPAAm‑1 displayed the strongest antimicrobial effect across both strains, with S. aureus showing greater sensitivity than E. coli. These findings highlight emulsion polymerisation as the preferred route for producing Fe₃O₄‑PNIPAAm with enhanced antibacterial properties.

Background

Magnetic thermoresponsive polymer nanocomposites, such as Fe₃O₄–PNIPAAm, combine magnetic responsiveness with the temperature‑sensitive LCST behavior of PNIPAAm (~32 °C). The magnetic core facilitates rapid separation under an external field, while the polymer shell offers colloidal stability and functionalisation sites for drugs or biomolecules. Applications span water treatment, drug delivery, magnetic resonance imaging contrast, and hyperthermia‐based cancer therapy. Three principal synthesis strategies are employed: (1) physical addition of pre‑formed Fe₃O₄ and PNIPAAm particles; (2) in‑situ precipitation of Fe₃O₄ within a PNIPAAm matrix; and (3) emulsion polymerisation of N‑isopropylacrylamide around Fe₃O₄. The choice of method influences particle size, surface chemistry, magnetisation, and, ultimately, biological performance. Understanding these effects is essential for designing nanocomposites with optimal stability and antibacterial efficacy.

Methods

Chemicals

All reagents (FeCl₃·6H₂O, FeCl₂·4H₂O, NH₄OH, N‑isopropyl‑acrylamide, BIS, SDS, APS) were obtained from Sigma‑Aldrich (Germany) and used without further purification.

PNIPAAm Synthesis (Emulsion Polymerisation)

NiPAM (4 g), BIS (0.2 g) and SDS (0.3 g) were dissolved in 350 mL deionised water at 70 °C under N₂. APS (0.0035 g) in 1 mL water initiated polymerisation; the reaction ran for 4 h, after which particles were washed and collected by centrifugation (12 000 rpm, 30 min).

Fe₃O₄ Nanoparticle Preparation

FeCl₂·4H₂O (1.9 g) and FeCl₃·6H₂O (5.4 g) (1:2 molar ratio) were dissolved in 100 mL DI water, heated to 70 °C, and precipitated with NH₄OH (6 mL). The black suspension was stirred for 30 min, washed repeatedly, and dried in a rotary evaporator (25 mbar, 40 °C).

Fe₃O₄‑PNIPAAm‑1 (Emulsion Polymerisation)

NiPAM (0.4 g), Fe₃O₄ (0.2 g), BIS (0.2 g), SDS (0.3 g) were mixed in 350 mL DI, heated to 70 °C under N₂, and polymerised with APS (0.0035 g). After 4 h, the composite was washed, centrifuged, and dried.

Fe₃O₄‑PNIPAAm‑2 (In‑Situ Precipitation)

FeCl₂ (0.148 g), FeCl₃ (0.4 g), and 10 mL DI were added to 1 g PNIPAAm. NH₄OH (3 mL) induced immediate black precipitation; the mixture was stirred at 70 °C for 30 min, washed, centrifuged, and dried.

Fe₃O₄‑PNIPAAm‑3 (Physical Addition)

1 g PNIPAAm, 0.5 g Fe₃O₄, and 5 mL DI were mixed and stirred at 70 °C for 30 min. The suspension was washed, centrifuged, and dried.

Characterisation

Size and zeta potential were measured by DLS after sonication. Thermogravimetric analysis (25–900 °C, 10 °C/min, N₂) quantified coating content. Magnetisation saturation was recorded with a vibrating sample magnetometer. SEM images (1.5 kV) revealed morphology. All measurements were performed in triplicate.

Bacterial Strains and Growth Conditions

E. coli CCM3954 (Gram‑negative) and S. aureus CCM3953 (Gram‑positive) were cultured overnight in soy‑nutrient broth before exposure to nanocomposites (0.01–1 g L⁻¹).

DNA Damage Assessment (Comet Assay)

Bacterial suspensions (10⁷ cells mL⁻¹) were incubated with 0.1 or 1 g L⁻¹ of each composite for 30 min at 37 °C. Standard comet protocol (lysis, electrophoresis, staining) was followed; 50 comets per sample were quantified using fluorescence microscopy (×400).

Growth, Viability, and Morphology

Growth rates were calculated from OD₆₀₀ measurements every 2 h over 6 h. EC₁₀ values were derived from inhibition percentages. Viability after 24 h was determined with a live/dead kit (excitation 485 nm, emission 528 nm for live; 645 nm for dead). Cell length (E. coli) and cluster area (S. aureus) were measured at ×600.

Statistical Analysis

Data were analysed by one‑way ANOVA followed by Dunnett’s test (GraphPad Prism). Significance was set at P < 0.05.

Results

SEM images confirmed that Fe₃O₄‑PNIPAAm‑1, –2, and –3 exhibited distinct morphologies, with Fe₃O₄‑PNIPAAm‑1 showing the narrowest size distribution and minimal agglomeration (Figure 1). TGA curves indicated that all composites were thermally stable above 400 °C, with Fe₃O₄‑PNIPAAm‑1 retaining 87 % of its mass, reflecting a high Fe₃O₄ loading (Figure 2). Zeta potentials were –15.6 mV (–1.58 mV for PNIPAAm), –16.4 mV (–1.8 mV for Fe₃O₄‑PNIPAAm‑3), confirming colloidal stability (Figure 3). Magnetisation saturation values were 50.4 emu g⁻¹ (Fe₃O₄‑PNIPAAm‑1), 53.7 emu g⁻¹ (Fe₃O₄‑PNIPAAm‑2), and 21.0 emu g⁻¹ (Fe₃O₄‑PNIPAAm‑3). DLS measurements at 25 °C and 45 °C revealed temperature‑dependent size changes consistent with LCST behaviour (Figure 4).
Comet assays showed significant DNA strand breaks in both E. coli and S. aureus after 30 min exposure to all composites (P < 0.001) (Figure 5). Growth‑rate assays demonstrated that Fe₃O₄‑PNIPAAm‑1 and –2 strongly inhibited bacterial proliferation, whereas Fe₃O₄‑PNIPAAm‑3 had a modest effect (Figure 6). EC₁₀ values were lowest for Fe₃O₄‑PNIPAAm‑1, indicating the highest antibacterial potency. Viability assays revealed dose‑dependent increases in dead cells, with Fe₃O₄‑PNIPAAm‑1 achieving up to 32 % dead E. coli and 50 % dead S. aureus at 1 g L⁻¹ (Figure 7). Morphological analysis showed E. coli elongation and S. aureus clustering at higher concentrations, reflecting stress responses.

Discussion

The synthesis route markedly influences the physicochemical attributes of Fe₃O₄‑PNIPAAm nanocomposites. Emulsion polymerisation yields particles with optimal size homogeneity, surface charge, and magnetisation, facilitating rapid dispersion and strong antibacterial action. In‑situ precipitation offers comparable thermal stability but slightly higher aggregation, while physical addition results in the least desirable properties and weakest antimicrobial effect. The observed DNA damage likely arises from oxidative stress and membrane disruption induced by Fe₃O₄ cores, corroborated by previous reports of Fe‑based nanoparticle genotoxicity. The superior activity of Fe₃O₄‑PNIPAAm‑1 underscores the importance of controlled synthesis for biomedical applications such as antimicrobial coatings, wound dressings, and targeted drug delivery.

Conclusions

Fe₃O₄‑PNIPAAm nanocomposites prepared via emulsion polymerisation (Fe₃O₄‑PNIPAAm‑1) exhibit the most favorable combination of thermal stability, surface charge, magnetisation, and antibacterial efficacy against both E. coli and S. aureus. In‑situ precipitation (Fe₃O₄‑PNIPAAm‑2) and physical addition (Fe₃O₄‑PNIPAAm‑3) produce less stable and less potent composites. These findings support the use of emulsion polymerisation for generating Fe₃O₄‑PNIPAAm materials suitable for clinical and environmental antimicrobial applications.

Abbreviations

Fe₃O₄‑PNIPAAm

Magnetic poly(N‑isopropylacrylamide)

MNPs

Magnetite nanoparticles