Effect of Nanoparticles Type and Content on the Antimicrobial Activity of Magnetoelectric Polymer‐Based Composites
Joana Moreira, Margarida M. Fernandes, Vitor Correia, Daniela M. Correia, Carmen R Tubio, Vesna Lazic, Senentxu Lanceros‐Mendez
TL;DR
This paper explores how magnetoelectric nanocomposites with different nanoparticles can inhibit bacterial growth when stimulated by magnetic fields.
Contribution
The study introduces magnetoelectric nanocomposites that show enhanced antimicrobial activity under magnetic stimulation.
Findings
Nanocomposites with 20% CoFe2O4 nanoparticles showed significant inhibition of E. coli and S. aureus at 1 Hz magnetic stimulation.
Magnetic stimulation increased antimicrobial effectiveness compared to static conditions.
P(VDF-TrFE) nanocomposites with CFO nanoparticles are promising for antimicrobial surfaces in medical and public settings.
Abstract
Antimicrobial materials are essential for the development of coatings for high traffic surfaces to prevent the adhesion and proliferation of microorganisms, playing a crucial role in infection control. In this study, different magnetoelectric nanocomposites exhibiting antimicrobial activity upon magnetic stimulation were developed by solvent casting. The nanocomposites, composed of poly(vinylidene fluoride‐co‐trifluoroethylene) [P(VDF‐TrFE)] with different contents (10 and 20% wt) of CoFe2O4 (CFO) or Fe3O4 nanoparticles, were developed to respond to a variable magnetic field, mechanically stimulating the piezoelectric component of the material and inducing surface potential variations. The antimicrobial properties of these materials were evaluated by exposing them to different magnetic frequencies (0.3 and 1 Hz) in a custom‐made magnetic bioreactor. The growth of Escherichia coli (E.…
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FIGURE 8- —Fundação para a Ciência e a Tecnologia10.13039/501100001871
- —Portuguese Foundation for Science and Technology
- —B‐EAMS
- —J.M. grant
- —Ministry of Science, Technological Development, and Innovation of the Republic of Serbia
- —European Union10.13039/501100000780
- —University of Burgos, and SAT of the University of La Laguna, Spain
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Taxonomy
TopicsAdvanced Sensor and Energy Harvesting Materials · Multiferroics and related materials · Nanoparticles: synthesis and applications
Introduction
1
The rapid increase in antimicrobial resistance (AMR) is a pressing concern for the future of global health and contemporary healthcare [1]. Each year, approximately 7.7 million deaths are associated to bacterial infections, with 4.95 million linked to drug‐resistant pathogens, while 1.27 million are caused by bacteria resistant to available antibiotics [2].
Aiming to address this growing health issue, antimicrobial materials have gained considerable attention in both industry and healthcare for their potential to protect surfaces, particularly high‐traffic surfaces (e.g, touch paths or touch screens) and medical devices. These materials prevent microbial adhesion and biofilm formation, helping to reduce the spread of harmful microorganisms in clinical settings and public spaces [3]. Among the different approaches, smart materials based on piezoelectric polymers like poly(vinylidene fluoride‐co‐trifluoroethylene) [P(VDF‐TrFE)] have been explored for their antimicrobial potential when exposed to physical stimuli [4, 5]. By applying mechanical cues, these polymers create varying electroactive microenvironments on their surface, which have been shown to effectively prevent bacterial adhesion and mitigate the development of bacterial resistance [6]. The electric field generated by the piezoelectric material can attract the negatively charged peptidoglycan layers in bacteria, and this interaction can lead to several effects, such as membrane disruption, as reported in [4].
In addition to mechanical cues, electroactive microenvironments can also be induced and modulated magnetically when using magnetoelectric materials [7]. The magnetoelectric effect occurs when a magnetic field induces changes in electrical polarization, or when an electric field induces magnetization variations in the material [5, 8]. To obtain such materials, piezoelectric polymers can be combined with magnetic or magnetostrictive nanostructures [7, 9]. Magnetoelectric composites have already demonstrated potential in biomedical applications, in particular for tissue regeneration, where the generate dynamic microenvironments allow for improved adhesion and proliferation of preosteoblasts [10]. Notable examples include nanocomposites that combine the piezoelectric P(VDF‐TrFE) polymer with magnetostrictive cobalt ferrite (CFO) or magnetic iron oxide (Fe_3_O_4_) nanoparticles (NPs) in biomedical studies [11, 12, 13]. Thus, it has been demonstrated that magnetoelectric films composed of P(VDF‐TrFE) polymer and CFO NPs enhance cell proliferation and differentiation under magnetic stimulation [12].
CFO and Fe_3_O_4_ NPs have also been explored for antimicrobial applications, showing potential to play a relevant role in this field. Studies report that both NPs exhibit biocidal properties, making them promise in this context [14, 15, 16]. In addition, CFO and Fe_3_O_4_ have been reported due to their favorable magnetoelectric properties and for allowing to obtain of biocompatible composites [12, 17, 18]. CFO exhibits a high magnetostrictive coefficient, which, when coupled with a piezoelectric matrix, allows efficient generation of local electric fields under a magnetic stimulus [14]. Fe_3_O_4_, while slightly less magnetostrictive, is widely reported as biocompatible [13]. These characteristics make both NPs particularly suitable for evaluating antimicrobial activity under magnetoelectric stimulation. Compared to alternatives such as Terfenol‐D or NiFe_2_O_4_, CFO and Fe_3_O_4_ offer a better balance between magnetoelectric response, stability in biological environments, and low cytotoxicity, which is critical for potential in vivo applications [19, 20, 21].
The incorporation of CFO NPs into cellulose/polyaniline polymer matrices also exhibits good antimicrobial activity against E. coli, Bacillus subtilis, and Candida albicans as reported in [22]. Similarly, the inclusion of Fe_3_O_4_ NPs in chitosan‐pectin polymer films exhibited ferrimagnetic behavior, increased dielectric constant in increased NPs concentration, and enhanced antimicrobial activity against E. coli and Staphylococcus epidermidis (S. epidermidis) [23]. Therefore, incorporating these NPs into piezoelectric matrices may enhance the antimicrobial effect by leveraging the synergy between their biocidal action and the electroactive microenvironments generated by the piezoelectric polymers. Piezoelectric‐based materials that generate antimicrobial effects by creating electroactive microenvironments in response to magnetic stimuli still represent an emerging and innovative strategy for enhancing antimicrobial properties [24]. The literature reports that nanocomposites developed from the piezoelectric polyvinylidene fluoride (PVDF) polymer, filled with nickel nanowires, exhibit more than 55% bacterial growth inhibition under controlled dynamic magnetic stimulation [7]. This approach is particularly appealing because it enables to take advantage of mechanical (piezoelectric effect) or magnetic (magnetoelectric) variations of the environment to stimulate the surface, offering a chemical‐free, physically‐based method for achieving antimicrobial effects [25].
This study proposes the development of a piezoelectric polymer P(VDF‐TrFE)‐based magnetoelectric polymer nanocomposite incorporating different contents of magnetostrictive CFO or magnetic Fe_3_O_4_ NPs, selected according to their magnetoelectric response and biosafety concentrations. The main goal is to quantify the antimicrobial and/or antibiofilm effect as a function of nanoparticle type and loading under magnetic stimulation. While previous studies have explored magnetoelectric systems for antimicrobial applications [21, 26], the present work differentiates itself by systematically evaluating how nanoparticle type (CFO vs. Fe_3_O_4_) and concentration influence both the magnetoelectric response of P(VDF‐TrFE) nanocomposites and their corresponding biological activity. This approach enables a direct correlation between particle composition, magnetoelectric performance, and antimicrobial efficacy. Furthermore, by employing biosafe concentrations and low‐frequency magnetic stimulation that mimics realistic environmental conditions, our study provides a safe and practical strategy for remote activation. Altogether, these properties highlight the potential of the proposed system as a promising platform for the development of antiseptic materials suitable for hospital settings and public spaces.
Materials and Methods
2
Materials
2.1
N,N‐dimethylformamide (DMF, pure grade) was obtained from Merck, and poly(vinylidene fluoride‐co‐trifluoroethylene), P(VDF‐TrFE), 70/30 from PiezoTech (Lyon, France). All the reagents used for the magnetite synthesis (Fe_3_O_4_) were analytical grade and used without further purification. Ferric chloride (FeCl_3_) and sodium hydroxide (NaOH) were purchased from Merck, Germany.
Cobalt ferrite, CFO, NPs (M_w_ = 234.62 g mol^−1^) with 35–55 nm size range and saturation magnetization M_s_ = 46.8 emu/g were obtained from Nanoamor (Katy, TX, USA).
Synthesis and Characterization of Magnetite Nanoparticles
2.2
For the synthesis of magnetite NPs, stock aqueous solutions of FeCl_3_ (dissolved in 2 mol dm^−3^ HCl) and Na_2_SO_3_, with concentrations of 2 and 1 mol dm^−3^, respectively, were first prepared. Concentrated aqueous ammonia (NH_3_) was diluted before use.
The precipitation procedure was followed according to the method presented in [27]. Briefly, stock solutions of FeCl_3_ and Na_2_SO_3_ were mixed under stirring, leading to a color change indicative of complex formation. This mixture was then rapidly added to a diluted ammonia solution, resulting in the formation of a black precipitate. The suspension was stirred for 30 min, and the precipitate was separated using a permanent magnet. The product was washed several times with deionized water to remove residual ions and then dried at room temperature.
Transmission electron microscopy (TEM) was performed to analyze the synthesized Fe_3_O_4_ NPs using a JEOL JEM‐2100 LaB6 instrument operated at 200 kV. The synthesized NPs show a saturation magnetization of 45 emu/g.
Processing of Magnetic Polymer Composites
2.3
P(VDF‐TrFE) was dissolved in DMF at a concentration of 15% (w/v). The solution was magnetically stirred at 30 °C, during approximately 2 h, until the polymer was completely dissolved.
For the development of P(VDF‐TrFE)/CFO and P(VDF‐TrFE)/Fe_3_O_4_ nanocomposites, the CFO and Fe_3_O_4_ NPs were initially sonicated in DMF at 10% (w/w_polymer_) and 20% (w/w_polymer_) for ≈30 min using an ultrasonic bath (FB15056, Fisherbrand, Massachusetts, USA) to ensure good dispersion. Next, a 15% (w/v) P(VDF‐TrFE) solution was added to the NPs dispersions and mechanically stirred until the solution was completely homogeneous. The NPs concentrations were selected based on studies indicating their safety for mammalian cells in cytotoxicity tests and suitable mechanical and magnetoelectric properties into the polymer matrix [13, 28].
To produce the neat P(VDF‐TrFE) films and the P(VDF‐TrFE)/CFO and P(VDF‐TrFE)/Fe_3_O_4_ nanocomposite films, the different solutions were poured onto a glass substrate and uniformly spread using an extender (doctor blade method) [29]. The glass substrates with the spread solutions were then placed in an oven (P‐Selecta) at 210 °C for 10 min to facilitate rapid solvent evaporation (Figure 1a). Although this temperature is above the melting point of P(VDF‐TrFE), the short treatment time prevents polymer degradation and has been shown to yield uniform films without affecting the β‐phase structure [4, 29, 30]. The preservation of the polymer's structure was further confirmed by FTIR analysis (Figure 4a).
Schematic presentation of the (a) experimental procedure used for the preparation of composites by the doctor blade method and (b) magnetic bioreactor used for the dynamic stimulation of the films in the antimicrobial tests.
After cooling to room temperature, the produced films showed an average thickness of 7 ± 0.03 µm, 10 ± 0,01 µm, 9 ± 0,01 µm, 6 ± 0,01 µm and 9 ± 0,01 µm for P(VDF‐TrFE) film, P(VDF‐TrFE)/CFO 10 wt.%, P(VDF‐TrFE)/CFO 20 wt.%, P(VDF‐TrFE)/Fe_3_O_4_ 10 wt.% and P(VDF‐TrFE)/Fe_3_O_4_ 20 wt.%, respectively.
Characterization of the Polymer Films
2.4
The surface and cross‐section morphology of neat P(VDF‐TrFE) film and P(VDF‐TrFE) films with CFO and Fe_3_O_4_ NPs was analysed by Scanning Electron Microscopy (SEM) using a Hitachi S‐4800 coupled with energy dispersive X‐ray spectroscopy (EDX), after the deposition of a conductive layer of sputtered gold/palladium. This microscope operated at an acceleration voltage of 5 kV, and samples were measured at a working distance of 7 mm and 500x magnification. The cross‐sections of polymer films were prepared by liquid nitrogen cryogenic fracture.
The polymer phase was determined by Fourier Transform Infrared Spectroscopy (FTIR). Measurements were performed in a Jasco FT‐IR‐4100 apparatus in attenuated total reflection (ATR) mode from 4000 to 600 cm^−1^.
Differential scanning calorimetry (DSC) measurements were performed in a Mettler Toledo 822e from 40°C to 220°C at a scanning rate of 10°C min^−1^ under nitrogen atmosphere. The degree of crystallinity (X_C_) of the samples was calculated using Equation (1):
where ΔH_m_ is the area of the melting peak of the sample and ΔH_m100_ the enthalpy of a 100% crystalline sample (103.45 J g^−1^) [31].
Thermogravimetry analysis (TGA) was performed using a TA Instruments Trios V5.8.0.4. The measurements were conducted over the temperature range from 25°C to 700°C, at a heating rate of 10 °C·min ^−1^ under a nitrogen atmosphere.
The magnetic properties of the nanocomposite samples were evaluated at room temperature using a MicroSense EZ7 vibrating sample magnetometer (VSM) over a magnetic field range from −18.000 to 18.000 Oe.
The wettability of the materials was assessed by measuring the contact angle with the surface of the sample of 3 µL distilled water drop. The contact angle measurements were conducted at room temperature using the DataPhysics OCA 20 contact angle system. For each sample, the contact angle was determined three times at different locations, and the results were presented as the average value and the corresponding standard deviation.
Characterization of the Magneto‐Mechanical Stimulus
2.5
To measure mechanical deformation when subjected to magnetic stimulation, which varies between ∼ 0.2T and ∼ ‐0.2T applied by a homemade magnetic bioreactor [13, 32], a strain gauge model KFH‐3‐120‐C1‐11L3M3R (OMEGA) was adhered to the sample surface using a two‐component epoxy adhesive (LOCTITE Hysol 3425). Considering the sample thickness, a low mechanical response of the material is expected, based on the magnetic field to which it is subjected.
Thus, with the measurement of the variation in the resistance of the strain gauge ΔR, and applying Equation (2), where GF is the gauge factor of the sensor, the length along the measurement axis is L0, and R0 is the resistance of the undeformed gauge, the dimensional variation of the sample ΔL, can be calculated:
In order to measure the deformation produced by the variation of the magnetic field in the sample, with greater accuracy, the strain gauge was connected as the active arm in a Wheatstone bridge configuration (Figure 6a), where the differential output voltage of the bridge, proportional to the variation of the resistance of the strain gauge (and consequently, to the deformation) is given by Equation (3):
where V_+_ corresponds to the supply voltage of the Wheatstone bridge and R_0_ = R_1_ = R_2_ = R_3_ = 120Ω.
Further, the differential output voltage of the bridge, was connected to the input of an instrumentation amplifier model INA114 (Texas Instruments).
An external gain resistance (Rg) of 470 Ω was used to configure the amplification of the signal provided by the bridge, corresponding to a gain of approximately 107.4. The amplified output voltage was then measured using a Rigol DM3068 6 ½ digit benchtop multimeter, and the AC filter was activated. The multimeter was connected via USB interface to a computer, where a control application developed in Python, using instrument communication libraries (such as NI‐VISA, managed through National Instruments' NI MAX), was used for continuous recording of voltage data.
During the measurements, samples were kept in a 24‐well culture plate containing cell culture medium, positioned in the self‐built magnetic bioreactor [7, 13]. The samples were subjected to a variable magnetic field, generated by a linear displacement that travelled the distance between the centers of the plate wells, exposing them to a magnetic field cycle with amplitude between a negative maximum and a positive maximum.
The same procedure was performed using a sample without magnetic NPs, allowing to isolate the effect of the magnetic fillers on the sample response.
Antibacterial Activity
2.6
Escherichia coli K12 (E. coli), a Gram‐negative bacterium, and Staphylococcus aureus ATCC6538 (S. aureus), a Gram‐positive bacterium (purchased from American Type Culture Collection (LGC Standards S.L.U.), were used for the bacterial assays. The bacterial pre‐inoculum was prepared by transferring a single colony from the stock culture into nutrient broth (NB). This was incubated overnight at 37 °C with shaking at 200 rpm.
Bactericidal activity was assessed using a modified version of the standard shake flask method (ASTM‐E2149‐01). This method quantifies the reduction in the number of colonies formed, converted to the average colony‐forming units per milliliter (CFU mL^−1^) of buffer solution in the flask. Briefly, the pre‐inoculum bacterial cultures were centrifuged at 5000 rpm for 5 min, washed twice with 0.9% (w/v) NaCl solution at pH 6.5, and then adjusted to an optical density (OD) of 0.1 ± 0.01 at 600 nm using sterile saline solution (0.9% NaCl). This procedure resulted in a working inoculum of approximately 7.3 × 10⁷ CFU mL^−1^ for E. coli and 6.1 × 10⁹ CFU mL^−1^ for S. aureus.
Bacterial Viability Assay on Polymer Films
2.6.1
The films were cut into 13 mm circles and sterilized using UV light for a period of 30 min on each side. They were then placed at the bottom of a 24‐well plate. Following this, 500 µL of the working inoculum of E. coli and S. aureus was added to each well in contact with the material. Wells devoid of any material were used as controls for bacterial growth. For static conditions, the plate was incubated for 2 h at 37 °C without any external stimuli. For dynamic conditions, the plate was placed in the home made bioreactor system with magnetic stimulation for 2 h at 37 °C (Figure 1b) [32]. The bioreactor consists of a magnet positioned below each well of a 24‐well plate. The magnets are subjected to cyclic displacement at specific frequencies. This study applied a varying magnetic stimulation between ∼ 0.2 and ∼ ‐0.2T at two distinct frequencies: 0.3 and 1 Hz, with the permanent magnets below the culture wells moving up to 15 and 20 mm, respectively [10, 32]. The 1 Hz frequency was selected in order to investigate whether increasing the stimulation frequency could induce a more intense magnetoelectric effect, potentially enhancing the antimicrobial activity compared to that reported in the literature for a frequency of 0.3 Hz [7].
To evaluate the antimicrobial properties of the materials after incubation, a colony‐forming unit (CFU) assay was performed to assess the viability of bacterial cells in suspension after contact with the material under static and dynamic conditions. For the CFU assay, bacterial cultures were serially diluted in 0.9% NaCl (pH 6). Then, 20 µL of the solution for each dilution was plated onto NB agar plates and incubated at 37 °C for 24 h to quantify viable bacteria. The results were expressed as the average colony‐forming units (CFU mL^−1^) of the pre‐inoculum solution. Relative bacterial cell viability (%) was then determined for each condition and compared to cells incubated without any material or external stimulus.
A LIVE/DEAD BacLight Bacterial Viability Kit for microscopy (Invitrogen, US) was used to study bacterial adhesion to the surface of the films after incubation with and without applying of a magnetic field. The samples were stained with a mixture of 1.5 µL of green‐fluorescent SYTO 9 and 1.5 µL of red‐fluorescent propidium iodide in PBS for 15 min in the absence of light. Finally, the samples were imaged using a fluorescence microscope (Olympus BX63F2), and representative fields were captured using a magnification of 100x.
Bacterial Adhesion on Polymer Films
2.6.2
The ability of antimicrobial agents to combat biofilm‐associated infections is primarily determined by their impact on biofilm formation. After 24 h of incubation at 37 °C with the working inoculum of E. coli and S. aureus under static and dynamic conditions, the liquid medium was removed. The biofilms were washed three times with distilled water to remove non‐adherent bacteria, and a colorimetric crystal violet assay was performed to evaluate the total biomass on the surface, including viable and non‐viable bacterial cells and the extracellular matrix.
After washing the biofilm, the 24‐well plate was incubated for 120 min at 37 °C to fix the biofilm. Subsequently, 700 µL of 0.1% (w/v) crystal violet solution (Sigma–Aldrich) was added to each well and incubated at room temperature for 10 min. After discarding the crystal violet, the samples were washed three times with sterile distilled water. To elute the remaining stain, 500 µL of 30% (v/v) acetic acid (Sigma–Aldrich) was added, and the total biomass was quantified by measuring the absorbance at 595 nm. In all experiments, wells without material were used as positive controls.
Statistical Analysis
2.6.3
The results are presented as the means of the individual measurements with the corresponding standard deviations and were analysed using GraphPad Prism version 9.0.0 for Windows (GraphPad Software, San Diego, CA). One‐way analysis of variance (ANOVA) was used to determine statistical significance, followed by an unpaired two‐tailed Student's t‐test.
Results and Discussion
3
Morphological Analysis of Nanoparticles and Composites
3.1
A comprehensive morphological examination of the Fe_3_O_4_ NPs was carried out using TEM. Low‐magnification images (Figure 2) revealed that the sample primarily comprised spherical particles with diameters near 10 nm, alongside elongated rod‐like nanostructures approximately 100 nm long and 2 nm wide. The simultaneous formation of both morphologies can be attributed to the type of iron precursor used, the nature of the surfactants, and the solvent environment employed [33, 34].
High‐magnification TEM image of (a, b) magnetite NPs and (c) cobalt ferrite NPs.
On the other hand, the commercial CFO NPs, according to supplier information and previous studies, are spherical and are polydisperse with a size range from 35 to 55 nm (Figure 2) [35, 36].
The surface and cross‐section morphology of the neat P(VDF‐TrFE) films and nanocomposite samples was evaluated by SEM images (Figure 3). Independently of the filler type and content, the samples exhibit a compact and homogeneous surface, associated to the polymer crystallization by cooling to room temperature after solvent evaporation at 210°C, above polymer melting temperature [29]. Further, no large NPs aggregates are observed, indicating a suitable dispersion of the fillers.
SEM images of the different P(VDF‐TrFE) based samples in surface and cross section (inset): (a) Neat P(VDF‐TrFE) film; (b) P(VDF‐TrFE)/CFO 10 wt.%; (c) P(VDF‐TrFE)/CFO 20 wt.%; (d) P(VDF‐TrFE)/Fe3O4 10 wt.% and (e) P(VDF‐TrFE)/ Fe3O4 20 wt.% films. EDX images showing the presence and distribution of Co (green), Fe (yellow), and O (purple). The scale bars denote 200 µm for top view and 10 µm for cross‐section images (inset) and EDX images.
The incorporation of CFO and Fe_3_O_4_ NPs within the nanocomposite films is confirmed by EDX analysis. Furthermore, the EDX images reveal that the density of nanoparticles within the composite increases with increasing NPs concentration (Figure 3b–e).
Vibration Spectra, Thermal Analysis, Magnetic Properties and Wettability
3.2
The infrared spectra of neat P(VDF‐TrFE), P(VDF‐TrFE)/CFO, and P(VDF‐TrFE)/Fe_3_O_4_ films are shown in Figure 4a. Independently of the filler type and concentration, all samples exhibit the characteristic vibration modes of P(VDF‐TrFE) at 840, 886, and 1402 cm^− 1^ [37, 38]. These vibration modes indicate the crystallization of the polymers in the highly polar trans TTT’ β‐phase chain conformation [39]. Thus, the inclusion of CFO or Fe_3_O_4_ NPs does not alter the chain conformation of the P(VDF‐TrFE) polymer matrix or introduce new chemical interactions between the NPs and the polymer. Although the characteristic absorption bands at 840, 886, and 1402 cm^−1^ are typically assigned to the β‐phase of P(VDF‐TrFE), it should be noted that FTIR analysis alone cannot unambiguously distinguish between the β and γ phases, as their vibrational features may overlap. Complementary techniques such as X‐ray diffraction (XRD) would be necessary to confirm the crystalline phase structure more conclusively.
Neat P(VDF‐TrFE) films, P(VDF‐TrFE)/CFO 10 wt.%, P(VDF‐TrFE)/CFO 20 wt.%, P(VDF‐TrFE)/Fe3O4 10 wt.% and and P(VDF‐TrFE)/ Fe3O4 20 wt.% nanocomposites: a) FTIR‐ATR spectra, b) DSC thermograms, c) TGA curves, d) room temperature magnetic hysteresis curves, and e) surface wettability.
Additionally, the analysis of the thermal properties was performed to assess the transition temperatures, the degree of crystallinity (DSC), and the thermal stability (TGA). Two endothermic peaks, at approximately 107°C and 150°C, are observed in the DSC thermograms of the samples (Figure 4b). The first peak (T_fp_) at a lower temperature (107°C), corresponds to the ferroelectric‐paraelectric phase transition, while the second peak (T_m_) at 150°C is assigned to the melting temperature [40].
The degree of crystallinity of the different samples was calculated using Equation (1). It is shown that the degree of crystallinity decreases as the NPs content increases in the P(VDF‐TrFE) matrix (30%, 25%, 21% 23% and 20% for neat polymer, P(VDF‐TrFE)/CFO 10 wt.%, P(VDF‐TrFE)/CFO 20 wt.%, P(VDF‐TrFE)/ Fe_3_O_4_ 10 wt.%, and P(VDF‐TrFE)/ Fe_3_O_4_ 20 wt.%, respectively). The lower degree of crystallinity in the presence of NPs indicates that both CFO and Fe_3_O_4_ NPs act as defects during the crystallization process, also hindering spherulite growth and leading to defective ill‐crystallized structures [35, 41].
The thermal degradation of the samples was evaluated by TGA measurements. As shown in Figure 4c, independently of the NPs filler type and content, the degradation process is similar to neat P(VDF‐TrFE) polymer and occurs in one step. The degradation starts at ≈ 410°C for the neat polymer and ≈ 440°C in the presence of CFO and Fe_3_O_4_ NPs. Relatively to the neat polymer and the polymer in the presence of CFO and Fe_3_O_4_ NPs, the onset peak temperature does not show significant differences between samples for both composite types.
The quantification of the magnetic NPs content of the P(VDF‐TrFE) nanocomposites was assessed by VSM. Figure 4d shows the magnetization curves of the different P(VDF‐TrFE) nanocomposites determined at room temperature. Typically, CFO NPs present a maximum magnetization of 43 emu·g^−1^, at ≈5000 Oe applied magnetic field [41, 42]. Figure 4d shows that the saturation magnetization of the loops increases with the increase of CFO NPs in the polymer matrix due to the increase of magnetic filler content in the nanocomposite, reaching a maximum magnetization saturation of 5 and 11 emu·g^−1^ for the nanocomposites incorporating 10 and 20 wt.% of CFO NPs, respectively for an applied magnetic field of −18.000 and 18.000 Oe. On the other hand, the remanence remains constant, independently of the filler content, as a consequence of the good filler distribution. With respect to the Fe_3_O_4_ composites, Figure 4d, shows a complete absence of hysteresis, remanence, and coercivity, exhibiting a maximum magnetization of 2 and 7 emu·g^−1^ for the nanocomposites incorporating 10 and 20 wt.% of Fe_3_O_4_ NPs, respectively for an applied magnetic field of −18.000 and 18.000 Oe. For the Fe_3_O_4_ NPs, the room temperature is above the blocking temperature, and the magnetic moment of the particle is free to rotate in response to the applied magnetic field [8]. This superparamagnetic behavior is particularly advantageous for the magnetoelectric response. The absence of hysteresis ensures that the magnetically induced strain in the polymer matrix is proportional and repeatable, maximizing the efficiency of the magnetoelectric effect without energy loss or non‐linearities that would be present in materials with significant coercivity [43, 44].
Once surface wettability (hydrophobicity/hydrophilicity) is an important parameter that affects microbial adhesion and, consequently, microbial dissemination, the wettability of the different samples was assessed using the sessile drop technique (Figure 4e). All samples show contact angles ≈90°, presenting a moderate hydrophobic behaviour, which is in agreement with the literature [4, 45]. The introduction of CFO and Fe_3_O_4_ NPs slightly increases the contact angle comparatively to the P(VDF‐TrFE) film without NPs. This effect can be attributed to the increase in surface roughness induced by the NPs [12, 46, 47].
Magnetic Stimulus and Electromechanical Analyses
3.3
Since the materials developed in this study are magnetoelectric, they are expected to exhibit surface voltage variations in response to interaction with varying magnetic fields, due to the magneto‐mechanical response of the NPs and their coupling with the piezoelectric polymer [9]. This will in turn affect the behaviour of bacterial cells [5]. Therefore, evaluating the mechanical response of nanocomposites is crucial. Given the dynamic nature of the magnetic stimuli applied via the magnetic bioreactor, a corresponding mechanical response is expected in the samples [13, 14, 42].
According to the sensor technical information, its calibration factor is approximately 2.105 +/– 0.5% and its length along the measurement axis is L_0_ = 1.52 mm, with a resistance (R_0_) in the undeformed gauge state of 120Ω.
Through the electronic conditioning circuit (Figure 5a), the corresponding conversion expressions (section 2.5), and considering the total area of the sample, it is possible to obtain an approximation of the mechanical deformation to which the sample is subjected by the variation of the oscillatory magnetic field (Figure 5b).
(a) Schematic representation of the magnetic stimulus operating method and the measurement method applied to determine the electromechanical variation of the samples. (b) Evaluation of the deformation of the materials under a magnetic field with cyclic variation.
Figure 5b evidences the contribution of magnetic fillers as a function of magnetic material type and content. Thus, by considering the response of the samples without magnetic charge as a reference, a peak‐to‐peak variation larger than 300 µm is recorded for the samples with an active charge of 20% CFO, while a lower result was observed for the samples with an active charge of 10% Fe_3_O_4_ and 10% CFO, obtaining a peak‐to‐peak variation below to 100 µm.
In this way, a mechano‐mechanical input is provided that, considering the local piezoelectric nature of the polymer, will lead also to local voltage peak‐to‐peak variations of approximately 100, 200, 600 and 800 µV for 10% Fe_3_O_4_, 20% Fe_3_O_4_, 10% CFO, and 20% CFO, respectively. The local voltage variations were calculated according to Equation (4):
where ΔL is the dimension change, E Young's modulus, L_0_ is the initial length, d is the piezoelectric coefficient, t film thickness, ɛ the relative dielectric permittivity, and ɛ_0_ is free space permittivity.
Evaluation of Antimicrobial Activity Under Static and Dynamic Conditions
3.4
The antimicrobial activity of P(VDF‐TrFE) samples, with and without NPs (CFO and Fe_3_O_4_ NPs at concentrations of 10 and 20 wt.%), was evaluated in contact with Gram‐negative E. coli and Gram‐positive S. aureus. The study included the application of a variable magnetic field (Figure 1b) between ∼ 0.2 and ∼ ‐0.2T [7, 10, 14] at two different frequencies, 0.3 and 1 Hz, for 2 h, as previously described above.
Figure 6 shows the bactericidal effect (bacteria in suspension) against E. coli (Figure 6a) and S. aureus (Figure 6b) after contact with the materials under static and dynamic conditions.
Antimicrobial activity: (a) E. coli and (b) S. aureus after 2 h of incubation over the material under static and dynamic conditions. The dynamic conditions consisted in applying a magnetic field variation to the material at a stimuli frequency of 0.3 and 1 Hz. The calculated percentage of bacterial cell viability is related to the control cell of cells growing at the same conditions but without the material or stimuli. The results are the mean of three independent assays. ** P < 0.01, *** P < 0.001, **** P < 0.0001 when compared to each other and α P < 0.01, # P < 0.0001 when compared to the control sample P(VDF‐TrFE) under static conditions.
Under static conditions, independently of the filler type, an increase in NPs concentration leads to a reduction in bacterial viability, demonstrating their inherent antimicrobial activity in both bacterial strains. NPs are known to compromise bacterial cell membranes by disrupting their integrity, causing pore formation, leakage of cellular contents, and ultimately, cell death [48, 49]. Additionally, metal oxide NPs can generate, which damage proteins, nucleic acids, and lipids, further contributing to bacterial cell death [50, 51]. The presence of NPs can also interfere with metabolic processes, inhibit essential enzyme activity, and induce oxidative stress, leading to damage in critical cellular components such as DNA and proteins [52]. These results are consistent with the wettability behavior observed on the surface of the nanocomposites. The increase in hydrophobicity may contribute to reduced microbial adhesion, thereby hindering biofilm formation [53]. Comparing the two types of NPs, CFO NPs exhibit stronger antimicrobial activity than Fe_3_O_4_ NPs, resulting in lower bacterial viability for both strains. Studies have reported that CFO NPs possess significant antimicrobial properties, primarily due to the release of cobalt ions (Co^2^⁺), which disrupt enzyme functions and membrane integrity in bacteria [54]. In contrast, magnetite NPs are more inert in this respect and often require functionalization to enhance their antimicrobial efficacy [55, 56]. Furthermore, CFO NPs are more effective at generating ROS, exacerbating oxidative stress, and causing extensive damage to bacterial membranes and DNA. The combined effects of ROS generation and Co^2^⁺ release make CFO NPs more potent antimicrobial agents than Fe_3_O_4_ NPs [57, 58, 59, 60].
In contrast, under dynamic conditions, applying a magnetic field further decreases bacterial cell viability compared to static conditions, regardless of the presence of CFO and F_3_O_4_ NPs. When evaluating the influence of the magnetic field on control samples of neat P(VDF‐TrFE) film, a clear tendency for bacterial inhibition emerges due to the applied magnetic field (Figure 6). Although these films are non‐magnetic, minor responses under the applied field were observed, likely arising from small vibrations of the substrate or setup, which are then transduced by the piezoelectric nature of the polymer [61]. This effect may be explained by the direct effect of the magnetic field [62, 63]. These phenomena may cause alterations in the permeability of channels in the bacterial membrane [64, 65]. Further, the findings suggest that bacterial cell viability decreases with increasing magnetic field frequency. By increasing the stimulation frequency, the variation rate of the generated magnetic field also increases, leading to a larger direct effect on bacteria viability [21, 66].
Further, both E. coli and S. aureus were evaluated by comparing the effect of nanocomposite samples under static and dynamic conditions (0.3 and 1 Hz). The differences between the samples are statistically significant, being slightly more pronounced in the presence of magnetostrictive NPs under magnetic stimulation, particularly at a frequency of 1 Hz. These results demonstrate the relationship between magnetoelectric environments and magnetic NPs (mainly magnetostrictive CFO NPs), as a sharp decrease in bacterial cell viability was observed. The enhanced antimicrobial response observed in P(VDF‐TrFE) films containing CFO NPs under 1 Hz stimulation, compared to films with Fe_3_O_4_ NPs, can be attributed to a combination of factors. These include the superior magnetostrictive properties of CFO, improved magnetoelectric coupling with the piezoelectric matrix, greater generation of electric charges and ROS, and more intense mechanical interactions with bacterial cells (all of which are amplified by magnetoelectric stimulation) [18, 67]. Additionally, the larger size of CFO NPs (≈35–55 nm), in contrast to the smaller Fe_3_O_4_ NPs (≈10 nm), enables a more effective mechanical coupling with the polymer matrix, thereby enhancing the magnetostrictive response and contributing to a more potent antimicrobial effect [67, 68]. Collectively, these results demonstrate the ability of such magnetoelectric nanocomposites to inhibit bacterial growth, demonstrating the antimicrobial potential.
In Figure 7, Live/Dead fluorescent images of E. coli (Figure 7a.) and S. aureus (Figure 7b) are presented, where green‐fluorescent cells indicate viability, while red fluorescent cells signify compromised cell membranes. The effects observed on the material`s surface corroborate with the results obtained in Figure 6. Images of E. coli and S. aureus show that the number of adherent viable cells is more pronounced on the P(VDF‐TrFE) surface under static conditions compared to the same conditions with NPs incorporated into the material.
Fluorescence microscopy Live/Dead images of (a) E. coli and (b) S. aureus on different material surfaces and stimulation conditions after 2 h of incubation. The scale bar represents 20 µm for all images.
Furthermore, the biocide effect of the NPs is clearly visualized from the presence of a large number of compromised cells (red cells) for both E. coli and S. aureus, regardless of the applied conditions. An increase in NPs concentration leads to a corresponding increase in the number of compromised cells. On the other hand, when comparing the use of the two different NPs (CFO and Fe_3_O_4_ NPs), the ratio of compromised cells is slightly higher with CFO NPs. These results confirm that CFO NPs are more effective in antimicrobial applications. As previously discussed, it is reasonable to assume that CFO NPs are more reactive and consequently cause a higher number of cells with membrane damage, inhibiting cellular proliferation [69]. On the other hand, these results are consistent with the wettability analysis, which shows that films containing CFO exhibit a slightly higher contact angle, indicating increased hydrophobicity of the material. According to the literature, hydrophobic surfaces hinder bacterial adhesion, thereby inhibiting biofilm formation [70].
When dynamic magnetic fields are applied to the material, an increase in dead cells is observed, particularly in P(VDF‐TrFE)/CFO at 20 wt.%, where the electric surface charge variation is more pronounced (∼800 µV), indicating that the magnetoelectric environment enhances antibiofilm activity. Additionally, at higher magnetic frequencies, a slight reduction in bacterial cells occurs, which can be attributed to magnetomechanical effects and changes in the local electric field generated by the piezoelectric component of the nanocomposite [7, 71]. Finaly, these images highlight the potential to control bacterial growth inhibition and antibiofilm activity through the synergistic effects of the NPs, particularly CFO magnetostrictive NPs, and the electrical microenvironments created.
Biofilm Inhibition Under Static and Dynamic Conditions
3.5
The ability of P(VDF‐TrFE)/NPs composites surface in preventing biofilm formation was assessed after 24 h to evaluate their performance against E. coli and S. aureus (Figure 8). The reduction in total biomass was evaluated by quantifying crystal violet‐stained carbohydrates present in the biofilm`s extracellular matrix and cells [72]. Notably, the biomass reduction is negligible, reaching approximately only 10% in the P(VDF‐TrFE) samples under static conditions for both bacteria strains. Oppositely, when the samples are exposed to a polymer with CFO and Fe_3_O_4_ NPs, a higher biomass inhibition is observed. These findings align with the existing literature, which indicates that CFO and Fe_3_O_4_ NPs exhibit both biocidal and antifouling properties [73, 74, 75].
Total biomass quantification of (a) E. coli and (b) S. aureus after 24 h of incubation over the material under static and dynamic conditions. For dynamic conditions, a magnetic field variation was applied to the material at stimulus frequencies of 0.3 and 1 Hz. Bars represent mean ± SD from n = 3 measurements. ** P < 0.01, *** P < 0.001, **** P < 0.0001.
Further, in the presence of dynamic stimuli, mainly 1 Hz, the biomass inhibition increases ≈30% more for E. coli and 60% more for S. aureus. These results demonstrate that magnetoelectric effects and the resulting variable mechanical and electrical microenvironment can promote membrane rupture through interactions between the varying surface charge of the piezoelectric polymer and the negative charges on the bacterial membrane [76]. By combining the presence of NPs with magnetic stimuli, the biomass inhibition effect is enhanced due to the synergistic interaction between the NPs and the dynamic stimuli.
Finally, it is evident that biomass inhibition is more pronounced in S. aureus compared to E. coli. Generally, gram‐negative bacteria exhibit greater resistance due to the presence of lipopolysaccharides (LPS) in their outer membrane and the production of exopolysaccharides, which contribute to the formation of a dense, protective matrix that shields against antimicrobial agents [77, 78].
Conclusion
4
This study reports the development of electroactive P(VDF‐TrFE) films incorporating different types and contents of magnetic NPs and their potential applicability as antibacterial material.
Independent of the filler type and content, no significant changes were observed in both morphological and physical‐chemical properties of the polymer. The key innovation of this work lies in the synergistic use of magnetic NPs, particularly with a P(VDF‐TrFE) piezoelectric polymer to create magnetoelectric nanocomposites capable of converting external magnetic stimuli into localized mechanical and electrical responses at the material's surface. This new antimicrobial approach results in bacterial membrane disruption, enhanced ROS production, and increased antibiofilm activity, achieved without the use of chemical agents. Importantly, this approach was effective against both Gram‐positive (S. aureus) and Gram‐negative (E. coli) bacteria, demonstrating broad‐spectrum antibacterial action. Furthermore, the study shows that the antimicrobial effect was significantly intensified when the composites were exposed to low‐frequency magnetic fields (especially at 1 Hz), confirming that the dynamic magnetoelectric environment actively contributes to microbial inactivation. The larger size and magnetostrictive response of CFO NPs played a critical role in enhancing the magnetoelectric coupling and, consequently, the antimicrobial performance. Compared to other magnetic fillers such as Fe_3_O_4_, CFO NPs exhibit a stronger magneto‐mechanical response, which enhances the coupling with the piezoelectric matrix. This results in more efficient conversion of magnetic stimuli into electrical signals, which is key to promoting bacterial inactivation. This effect was observed primarily in films containing 20% CFO, which makes this material particularly promising for antimicrobial applications.
These findings position the developed nanocomposites as a novel class of non‐chemical, remotely activatable antibacterial coatings. Their ability to operate through external magnetic stimulation, or potentially respond to ambient electromagnetic fields, makes them highly versatile for real‐world applications. This includes integration into high‐touch surfaces in public spaces, hospital equipment, or medical devices, offering long‐term antimicrobial protection and reducing the risk of antibiotic resistance development. By leveraging physical mechanisms of action, this work opens new directions in the design of smart and responsive antibacterial materials.
Conflicts of Interest
The authors declare no conflict of interest.
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