Ammonia solution was added to the mixture of the iron salts (in bright yellow) and its color turned to black, resulting in the generation of iron (III) & iron (II) hydroxides because of the hydrolysis of Fe3+ and Fe2+, respectively. In addition, iron (III) hydroxide was converted to another compound (FeOOH) that reacted with Fe(OH)2 to produce Fe3O4. Fe2+ to Fe3+ molar ratio was 1:2 to obtain high efficiency in magnetite production and to prevent the oxidation of Fe2+ to Fe3+. General reactions in the process of Fe3O4 formation are listed below (Eqs. (1–4)) [45].

$$^\left(aq\right)+3^(aq)\to Fe(OH_(aq)$$

(1)

$$_\left(aq\right)\to FeOOH\left(aq\right)+_O\left(l\right)$$

(2)

$$F^\left(aq\right)+2^(aq)\to _(aq)$$

(3)

$$2FeOOH\left(aq\right)+Fe(OH_\left(aq\right)\to __\left(s\right)+2_O(l)$$

(4)

TEM images of oleic acid-coated magnetite nanoparticles are demonstrated in Fig. 2a, b. These images indicate that near-spherical nanoparticles with uniform size (approximately 10 nm) have been synthesized. It is also evident that the surface modification of nanoparticles with oleic acid did not meaningfully change the size of the nanoparticles. DLS analysis, which is based on the Brownian motion of the particles, provides quantitative results of the particle size and particle size distribution. Figure 2c illustrates a narrow size distribution in the range of 7.60–25.55 nm with an average of 11.8 nm, that is in good agreement with the TEM results.

a, b TEM images of magnetic nanoparticles: a 100 nm, b 50 nm; c The size distribution and the average size of the modified IONPs

Figure 3a indicates the X-ray diffractogram of IONPs. The XRD pattern of the nanoparticles exhibits peaks at 2Ɵ = 21.6, 35.3, 41.6, 50.7, 63.3, 67.5, and 74.5° which are attributed to (111), (220), (311), (400), (422), (511), and (440) planes, respectively. The results are in accordance with the magnetite (JCPDS 19–629) reference patterns [46, 47].

a XRD pattern; b FTIR spectrum; c TGA curve and b dTGA curve; e Magnetization properties of the IONPs

Figure 3b shows the FTIR spectrum of the modified IONPs. The characteristic bands at 444 and 590 cm−1 are assigned to Fe–O bonding [48]. Two characteristic bands at 1430 and 1524 cm−1 (due to stretching vibration of COO−), confirm the presence of oleic acid on the nanoparticle surface [49]. Other characteristic absorption peaks at 2920 and 2850 cm−1 are related to the asymmetric and symmetric stretching of CH2 in oleic acid structure, respectively. The bending vibration of the OH band can be attributed to the absorption of water molecules on the surface of the IONPs [46, 49].

TGA analysis was employed to ensure the surface modification of the IONPs with oleic acid. Figure 3c, d demonstrates the TGA and the corresponding derivative (dTGA) curves of the coated IONPs. The weight loss observed at temperatures below 150 °C was negligible, which is assigned to the evaporation of the adsorbed water. The weight loss in the second transition region (temperature range 210–280 °C) has occurred owing to the elimination of the free or physically adsorbed oleic acid molecules [50]. The other weight loss observed at 333 °C is associated with the degradation of the oleic acid covalently bound to the IONP surface [51].

Figure 3e illustrates the field-dependent magnetization (M–H) curves of the IONPs. Results revealed the reversible field-dependent magnetization curves with no hysteresis loops, coercivity and remanent magnetization, which demonstrates that the net magnetization of the IONPs is zero in the absence of EMF [52]. These results express the super-paramagnetic behavior of the synthesized IONPs with high saturation magnetization value of 48.97 emu/g [46].

Figures 4 and 5 show the FE-SEM image, EDX, and the element maps of Ag and the IONPs in the synthesized nanocomposites. EDX analysis confirmed the presence of Ag and the IONPs throughout the nanocomposites. According to the FE-SEM and the element map images, spherical nanoparticles are distributed uniformly among the fibers without any agglomerations.

FE-SEM images, EDX, and element maps of nanocomposites synthesized at the fixed IONPs concentration (10%) and different concentrations of AgNPs; a 0, b 0.05, c 0.5, d 1%

FE-SEM images, EDX, and element maps of the nanocomposites synthesized at the fixed AgNPs concentration (0.5%) and different concentrations of IONPs; a 0, b 5, c 10, d 15%

Figure 4a–d demonstrates the effect of four different concentrations of the AgNPs on the nanocomposites containing a fixed amount of the IONPs (10%). In Fig. 4a, C, O, and Fe elements can be identified in the maps. When silver nitrate was added to the composite, Ag also appeared in the results. Additionally, as shown in the element maps of Fig. 4a–d, higher concentrations of silver nitrate in the prepared solution leads to the increased content of AgNPs in the nanocomposite structure. The constant intensity of Fe peaks in EDX analysis, as well as the good distribution of this element in the maps (a-d), indicates uniform coating of the IONPs in samples 9, 10, 11, and 12. The same trend exists in Fig. 5a–d. The small diversity of Ag density in the element maps shows the fixed amount of AgNPs in samples 3, 7, 11, and 15 synthesized using a constant concentration of the silver nitrate in the precursor solution. In proportion to the IONPs concentration in the coating solution, the IONPs content in the composites increases.

Figure 6a demonstrates the X-ray diffractograms of GA/PVA/PCL/Ag and GA/PVA/PCL/Ag/Fe3O4 nanocomposites. The addition of the IONPs to the nanocomposites has reduced the intensity of the peaks at 2θ = 24.95 & 27.69°, due to the interaction between PCL and the IONPs [53]. The IONP-containing nanocomposites showed three new peaks at 2θ = 34.82, 41.51 and 67.69° (for (220), (311), and (511) planes). Moreover, the intensity of the peaks increases at 2θ = 50.53 & 74.79° due to (400) and (440) planes, respectively.

a XRD pattern, and b FTIR spectra of GA/PVA/PCL/Ag and GA/PVA/PCL/Ag/IO nanocomposites

FTIR spectra of GA/PVA/PCL/Ag and GA/PVA/PCL/Ag/Fe3O4 nanocomposites are shown in Fig. 6b. The main difference is the peak at 597 cm−1. This peak is related to the stretching vibration of the metal–oxygen absorption band (Fe–O bond), indicative of the presence of the magnetite nanoparticles. Additionally, interactions between the IONPs and the nanocomposites can change the intensities or the peak shift. In the presence of the IONPs, the peaks of the hydroxyl group, C–H (asymmetric and stretching), C=O stretching vibration, COO− symmetric stretching, and C–C stretching vibrations have been shifted to 3187, 2927, 1733, 1433, and 846 cm−1, respectively [54].

The magnetization–magnetic field (M–H) curves of the nanocomposites at 300 K are shown in Fig. 7. The zero coercivity and the reversible hysteresis behavior of the nanocomposites reveal their super-paramagnetic property, which occurs only at the nanoscale. The saturation magnetization value of the IONPs was 48.97 emu/g, which was reduced to 4.23, 6.98, and 11.16 emu/g for the nanocomposites containing three different concentrations of the IONPs (5, 10, and 15%, respectively). However, the result also exhibits super-paramagnetic property [52, 55, 56]. Results demonstrate higher saturation magnetization values compared to the other researchers' studies such as Ahn and Kang [57]. Using the coating method instead of merging the nanoparticles into the solution prepared for the electrospinning process, increases the particle size and improves the magnetic property since magnetic characterization depends on the size of the synthesized nanoparticles [58].

The magnetization properties of the IONPs-containing nanocomposites

The effects of AgNPs content, IONPs concentration, and applying EMF, on the antibacterial activity of the nanocomposites against different microbial strains (S. aureus and methicillin resistant Staphylococcus aureus (MRSA) (Gram-positive), P. aeruginosa and E. coli (Gram-negative), and C. albicans (Yeast)), were investigated using two methods of disk diffusion and colony counting. Due to a large number of samples, only 5 samples include sample 1 (control sample), sample 4 (containing the highest amount of silver nanoparticles), sample 13 (containing the highest amount of iron oxide nanoparticles), sample 6 (containing the lowest amount of both silver nanoparticles and iron oxide) and Sample 16 (containing the highest amount of both silver nanoparticles and iron oxide) were used for microbial strains of MRSA, E. coli, and C. albicans.

According to Fig. 8a, b, the nanocomposites containing 0.05% AgNPs show low antibacterial activity. By increasing the concentration of AgNPs (to 1%), the diameter of the inhibition zone increases since more AgNPs can be released and higher antibacterial activity can be obtained (Figs. 9a, b, 10a, b, 11a, b) [59, 60]. AgNPs adhesion to the cell wall through electrostatic interactions causes its damage. In the following, it penetrates the cell and pushes the cell content out, which results in DNA deformation and disrupting its transcription and translation, and the generation of reactive oxygen species (ROS) and free radicals [13, 61].

Antibacterial activity of IONPs-free nanocomposites containing 0, 0.05, 0.5 and 1% AgNPs (samples 1, 2, 3 and 4, respectively) against a Staphylococcus aureus, and b Pseudomonas aeruginosa

Antibacterial activity of Ag/IO nanocomposites against MRSA, a and c in the absence and b and d in the presence of EMF

Antibacterial activity of Ag/IO nanocomposites against E.Coli, a and c in the absence and b and d in the presence of EMF

Antibacterial activity of Ag/IO nanocomposites against C. Albicans, a and c in the absence and b and d in the presence of EMF

The content of IONPs in the nanocomposite is another influential factor affecting antibacterial activity. Figures 9a, b, 10a, b, 11a, b, 12a, b shows that increasing the IONPs content from 5 to 15% leads to increased antibacterial activity. The main mechanism of the antibacterial activity of IONPs is the oxidative stress caused by ROS. ROS involve superoxide radicals (O2−), hydroxyl radicals (–OH), singlet oxygen (1O2), and hydrogen peroxide (H2O2), which penetrate the bacteria cells and damage their proteins and DNA [62,63,64]. On the other hand, bacteria use adhesive surface structures to attach to tissues. The attachment of IONPs to the cell wall and surface structures of bacteria causes the bacterial adherence factors to be occupied and inactivated, preventing them from binding [65].

Antibacterial test results of AgNPs-free nanocomposites containing 0, 5, 10 and 15% IONPs (samples 1, 5, 9, and 13, respectively) against Staphylococcus aureus, a in the absence and b in the presence of EMF

A comparison of the images in Figs. 9c, d, 10c, d, 11c, d, 13, 14 and the data in Tables 1, 2, 3, 4 and 5 demonstrates that the presence of EMF increases the antibacterial activity of IONPs-containing nanocomposites. The magnetic moments of the IONPs are randomly oriented in the absence of EMF. Therefore, net magnetization is zero. Applying a strong enough EMF forces the magnetic moments of the IONPs to align along the magnetic field direction [66].

Antibacterial activity of Ag/IO nanocomposites containing 0.05, 0.5, and 1% AgNPs; and 5% (a and b), 10% (c and d), and 15% IONPs (e and f) against S. aureus, (a, c and e) in the absence and (b, d, and f) in the presence of EMF

Antibacterial activity of Ag/IO nanocomposites containing 0.05, 0.5, and 1% AgNPs; and 5% (a and b), 10% (c and d), and 15% IONPs (e and f) against P. aeruginosa, (a, c and e) in the absence and (b, d, and f) in the presence of EMF

Due to the reasonable amount of the IONPs in the nanocomposites and their superparamagnetic properties, the applied EMF increases the IONPs release and improves the antibacterial activity. Moreover, the controlled release of IONPs leads to the more accessible release of the AgNPs, due to the increased porosity, and thus the better antibacterial activity of the nanocomposites [67, 68].

In addition to the disc diffusion assay, the antibacterial activity of the nanocomposites against two bacterial strains of S. aureus and P. aeruginosa was also carried out through the colony counting method, which is shown in Table 6.

From this table it is obvious that the number of bacterial colonies of petri dishes corresponding to nanocomposites containing silver and iron oxide nanoparticles for both pathogenic bacteria is significantly lower than the control nanocomposite (sample 1). The results show that the addition of silver nanoparticles (sample 4), the addition of iron oxide nanoparticles (sample 13), the simultaneous integration of both nanoparticles (samples 6 and 13), and the use of a magnetic field reduce the number of bacterial colonies. These results are in agreement with agar disk diffusion analysis.

Among the microbial strains, P.aeruginosa and MRSA showed the highest and lowest sensitivity to the antibacterial nanocomposites, respectively. The observed differences can be attributed to the presence of a thick layer of peptidoglycan in the cell wall of gram-positive bacteria, which acts like a barrier against the penetration of the antibacterial nanoparticles into the bacteria and affects their performance [39, 69].

ROS generation was measured by the DCFH-DA assay after exposing the nanocomposites to S. aureus and P. aeruginosa to ascertain whether it has an effect on the antibacterial mechanism of Ag/IO nanocomposites or not. The results revealed that NPs-containing nanocomposites, produced more ROS compared to untreated nanocomposite, in the presence of both S. aureus and P. aeruginosa in a concentration-dependent manner. Increasing the concentration of AgNPs to 1% leads to about 8% increase in ROS as compared to NPs-free nanocomposites. Similarly, bacteria exposed to IONPs-containing nanocomposites demonstrated increased ROS production (Fig. 15).

Effect of Ag/IO NPs-containing nanocomposites on ROS formation in presence of S. aureus and P. aeruginosa bacterial cells

Altogether, the above data suggest that AgNPs act as oxidative stress inducer agents and cause the loss of cell viability in S. aureus and P. aeruginosa bacteria through ROS generation. ROS formation of AgNPs-containing nanocomposites can be assigned to the higher silver ion release. The interaction between metal NPs and bacterial cells often leads to ROS production, which damage to proteins and nucleic acids. The IONPs also produce ROS at their surface and increasing the concentration, causes increased ROS formation as more NPs are released from the nanocomposites [39, 70]. Taken together, the above finding suggest that ROS formation is a possible mechanism responsible for bacterial cell death in presence of Ag/IO nanocomposites.

Cytotoxicity assay was carried out to study the biocompatibility of AgNPs, IONPs, and EMF on the fibroblast and macrophage cells. Cells were cultured on the nanocomposites for 1, 3, 5, and 7 days to perform the MTT assay. Generally, the type of nanoparticles, their shape, size, dose, surface properties, and the ways they are added to the system affect their toxicity [58]. According to Figs. 16a, b and 17, cells were able to grow on all nanocomposites; but increasing AgNPs and IONPs contents in the nanocomposites reduced cell proliferation due to the more released silver and iron oxide nanoparticles. Results revealed dose-dependent cytotoxicity of the silver nanoparticles [4, 16]. Increasing the AgNPs concentration to 1% reduced cell viability of fibroblasts and macrophages to about 80%. Ankamwar et al. showed that IONPs are not toxic in the concentration range of 0.1–10 µg/mL−1, but cell viability decreases when the concentration reaches 100 µg/mL−1 [