Silver Nanoparticles Grown on Cross-Linked Poly (Methacrylic Acid) Microspheres: Synthesis, Characterization, and Antifungal Activity Evaluation

: Silver nanoparticles (AgNPs) exert profound physicochemical, biological, and antimicrobial properties, therefore, they have been extensively studied for a variety of applications such as food packaging and cultural heritage protection. However, restrictions in their stability, aggregation phenomena, and toxicity limit their extensive use. Hence, the use of functional substrates that promote the silver nanoparticles’ growth and allow the formation of uniform-sized, evenly distributed, as well as stable nanoparticles, has been suggested. This study reports on the fabrication and the characterization of hydrophilic polymer spheres including nanoparticles with intrinsic antifungal properties. Poly (methacrylic acid) microspheres were synthesized, employing the distillation precipitation method, to provide monodisperse spherical substrates for the growth of silver nanoparticles, utilizing the co-precipitation of silver nitrate in aqueous media. The growth and the aggregation potential of the silver nanoparticles were studied, whereas the antifungal activity of the produced nanostructures was evaluated against the black mold-causing fungus Aspergillus niger . The produced structures exhibit dose-dependent antifungal activity. Therefore, they could potentially be employed for the protection and preservation of cultural heritage artifacts and considered as new agents for food protection from fungal contamination during storage.


Introduction
In recent years, nanotechnology has played a crucial role in food packaging improvement by combining antimicrobial agents with external barrier properties. Food deterioration reduction, resulting in prolonged food preservation, can be feasible through microbial growth retardation or inhibition via nanotechnology usage in packaging products [1,2]. The exploitation and incorporation of nanoparticles with antimicrobial properties into a variety of food contact materials such as papers, spheres, and fibers are a priority for studies aiming at food storage increment [3].
Another field of nanotechnology implementation is related to cultural heritage protection. Most of the tangible cultural heritage is predominantly based on archives (books, maps) and artwork (paintings, drawings, sculptures), usually utilizing paper, wood, or stone [4][5][6]. These materials are susceptible to degradation and biodeterioration by microorganisms, especially from fungi, since organic matter, like cellulose, is a source of essential nutrients [7]. Fungal growth on stone and wood constitutes a serious challenge as well [8].
determine [37]. Evidently, methodologies that employ substrates for the controlled growth of AgNPs without the use of a capping agent could potentially overcome these challenges, while still retaining their antimicrobial efficiency.
Herein, we report the preparation of cross-linked poly (methacrylic acid) (PMAA) microspheres for the growth of AgNPs. PMAA in aqueous media is almost entirely deprotonated and, therefore, can absorb and retain water, and it is accepted as an environmentally friendly chemical structure [38]. Consequently, PMAA is an ideal substrate for the growth of the AgNPs, since it has the potential to significantly contribute to the controlled oxidation of the AgNPs via the absorption of water, while effectively exploiting the association of fungal growth with high moisture levels [39,40]. In addition, the proposed methodology provides an alternative approach for producing stable, uncapped AgNPs. The uncapped surface of AgNPs has the potential to exploit the surface plasmon resonance of AgNPs, thus enabling a variety of sensing applications utilizing the surface-enhanced Raman spectroscopy (SERS) which is closely correlated with the coupling between the electric field of the incident beam and the field stimulated by the collective oscillation of valence electrons [37]. Chatzipavlidis et al. have previously reported the enhanced Raman signal in the detection of the Rhodamine B molecule as a potential imaging application whereas similar SERS methodology has been applied to identify Cr(VI) and organic pollutants, respectively, for environmental analysis applications [40][41][42][43]. The proposed methodology for AgNPs synthesis is an alternative to the standard procedure utilizing capping agents for the control over the nanoparticles' size and aggregation whereas it has the potential to provide the foundation for producing nanocomposite materials with tailored properties.
Infrared spectra (FT-IR) were recorded using a Cary 630 Agilent FTIR spectrometer equipped with a diamond ATR crystal, whereas a 670 UV-Vis spectrophotometer was used to measure the ultraviolet absorption spectra (UV-Vis) of the resulting structures. Scanning and transmittance electron microscopy (SEM, TEM) were employed to determine the size and shape of the synthesized microspheres, and energy dispersive X-ray spectroscopy (EDS) was used for the elemental analysis. The SEM and TEM were accomplished utilizing an FEI inspect microscope with a W (Tungsten) filament operating at 25 kV equipped with EDAX GENESIS (AMETEK PROCESS and ANALYTICAL INSTRUMENTS), and an FEI CM20 microscope operating at 200 kV, respectively. TEM samples were prepared by placing one drop of a diluted sample on a carbon-coated Cu grid and allowing the solvent to evaporate. Thermogravimetric analyses (TGA) were performed on a TGA Netch STA 449 Jupiter, whereas a Malvern Zetasizer Nano ZS apparatus was utilized for measuring the size, polydispersity, and surface charge of the polymer microsphere by dynamic light scattering (DLS).

Synthesis of P(MAA-co-MBA) Microspheres
For the synthesis of polymer microspheres, a modified version of the distillation precipitation polymerization procedure has been utilized as previously described [44,45]. Briefly, ACN, the monomer methacrylic acid, the cross-linker MBA, and the initiator AIBN were placed in a round-bottom flask and were vigorously stirred under an argon atmosphere. The concentration of the monomer was at 1% w/v (in relation to ACN, 350 mL), whereas the concentration of the cross-linker and the initiator were at 10% and 1.25% w/w, respectively (in relation to the monomer). After 10 min the mixture turned opalescent and then white, indicating the formation of polymer nanoparticles; at that point, the temperature was in-creased to 85 • C to initiate the distillation of the solvent and 15 mL of ACN was distilled. Centrifugation at 8000 rpm for 10 min and rinsing with distilled ACN was carried out to remove unreacted species.
2.3. Silver Nanoparticles' Growth on P(MAA-co-MBA) Microspheres (PMAA@Ag) The growth of AgNPs onto the surface of P(MAA-co-MBA) microspheres was accomplished utilizing the chemical co-precipitation of AgNO 3 in the presence of NaBH 4 as the reducing agent [30]. In a glass beaker 100 mL of deionized water, 100 mg of P(MAA-co-MBA) microspheres, and 50 µL of AgNO 3 (2N) were placed and stirred for varying time periods (1 to 24 h). Prior to the addition of the reducing agent, the colloidal solution was centrifuged (8000 rpm, 10 min) and redispersed in H 2 O to remove the free silver ions and the nitrate counter ions. Then, NaBH 4 (10 mM) was added dropwise into the mixture and the color gradually changed from white to yellow, to dark red, and then to light brown, depending on the stirring time of the P(MAA-co-MBA)/AgNO 3 solution, thus indicating the formation of the AgNPs. Figure 1 depicts a schematic representation of the growth of the AgNPs on P(MAA-co-MBA) microspheres (PMAA@Ag).
AIBN were placed in a round-bottom flask and were vigorously stirred under an argon atmosphere. The concentration of the monomer was at 1% w/v (in relation to ACN, 350 mL), whereas the concentration of the cross-linker and the initiator were at 10% and 1.25% w/w, respectively (in relation to the monomer). After 10 min the mixture turned opalescent and then white, indicating the formation of polymer nanoparticles; at that point, the temperature was increased to 85 °C to initiate the distillation of the solvent and 15 mL of ACN was distilled. Centrifugation at 8000 rpm for 10 min and rinsing with distilled ACN was carried out to remove unreacted species.

Silver Nanoparticles' Growth on P(MAA-co-MBA) Microspheres (PMAA@Ag)
The growth of AgNPs onto the surface of P(MAA-co-MBA) microspheres was accomplished utilizing the chemical co-precipitation of AgNO3 in the presence of NaBH4 as the reducing agent [30]. In a glass beaker 100 mL of deionized water, 100 mg of P(MAA-co-MBA) microspheres, and 50 μL of AgNO3 (2N) were placed and stirred for varying time periods (1 to 24 h). Prior to the addition of the reducing agent, the colloidal solution was centrifuged (8000 rpm, 10 min) and redispersed in H2O to remove the free silver ions and the nitrate counter ions. Then, NaBH4 (10mM) was added dropwise into the mixture and the color gradually changed from white to yellow, to dark red, and then to light brown, depending on the stirring time of the P(MAA-co-MBA)/AgNO3 solution, thus indicating the formation of the AgNPs. Figure 1 depicts a schematic representation of the growth of the AgNPs on P(MAA-co-MBA) microspheres (PMAA@Ag).

Evaluation of Antifungal Activity Using the Dilution Method
Fungal strain A. niger ATCC 16888, used in the antifungal activity evaluation assay, was kindly provided by Dr. Gonou-Zagou (Department of Biology, National and Kapodistrian University of Athens). Fungal spore suspensions were stored in 0.1% Tween and 20% glycerol at −80 °C prior to use. Before each assay, fungal cells were inoculated in PDA plates and grown at 27 °C for four days. Petri dishes (5.5 cm) were covered with PDA culture media in which increasing concentrations of PMAA@Ag in sterile distilled water were added. The selected final concentrations were 5, 1, and 0.5 mg PMAA@Ag/mL. A fungal inoculum (10 3 conidia per mL, in 0.1% Triton X-100) was added to each petri dish and the fungal growth was monitored over a positive growth control sample without the addition of any antifungal agent. Plates were incubated at 27 °C and the fungal growth was monitored for up to ten days.

Synthesis of PMAA-co-MBA Microspheres
Cross-linking of the polymer microsphere is vital for the spherical shape to be retained in aqueous media. FT-IR spectroscopy was utilized to qualitatively identify the cross-linking of the P(MAA-co-MBA) microspheres. Therefore, an additional sample of PMAA microspheres was prepared under the same conditions in the absence of the

Evaluation of Antifungal Activity Using the Dilution Method
Fungal strain A. niger ATCC 16888, used in the antifungal activity evaluation assay, was kindly provided by Dr. Gonou-Zagou (Department of Biology, National and Kapodistrian University of Athens). Fungal spore suspensions were stored in 0.1% Tween and 20% glycerol at −80 • C prior to use. Before each assay, fungal cells were inoculated in PDA plates and grown at 27 • C for four days. Petri dishes (5.5 cm) were covered with PDA culture media in which increasing concentrations of PMAA@Ag in sterile distilled water were added. The selected final concentrations were 5, 1, and 0.5 mg PMAA@Ag/mL. A fungal inoculum (10 3 conidia per mL, in 0.1% Triton X-100) was added to each petri dish and the fungal growth was monitored over a positive growth control sample without the addition of any antifungal agent. Plates were incubated at 27 • C and the fungal growth was monitored for up to ten days.

Synthesis of PMAA-co-MBA Microspheres
Cross-linking of the polymer microsphere is vital for the spherical shape to be retained in aqueous media. FT-IR spectroscopy was utilized to qualitatively identify the cross-linking of the P(MAA-co-MBA) microspheres. Therefore, an additional sample of PMAA microspheres was prepared under the same conditions in the absence of the crosslinker. As shown in Figure 2, it can be observed that the spectra of both materials exhibit a strong vibration at approximately 1700 cm −1 which is attributed to the carbonyl vibration, yet the IR spectrum of P(MAA-co-MBA) exhibits an additional peak at 1531 cm −1 , corresponding to the amide vibration of the cross-linker's molecule. Evidently, the two spectra exhibit identical vibrations with the exception of the vibration at 1531 cm −1 at the P(MAA-co-MBA) spectrum which corresponds to the amide bond vibration evidencing qualitatively the successful cross-linking of the PMAA polymer.
crosslinker. As shown in Figure 2, it can be observed that the spectra of both materials exhibit a strong vibration at approximately 1700 cm −1 which is attributed to the carbonyl vibration, yet the IR spectrum of P(MAA-co-MBA) exhibits an additional peak at 1531 cm −1 , corresponding to the amide vibration of the cross-linker's molecule. Evidently, the two spectra exhibit identical vibrations with the exception of the vibration at 1531 cm −1 at the P(MAA-co-MBA) spectrum which corresponds to the amide bond vibration evidencing qualitatively the successful cross-linking of the PMAA polymer.

Size Distribution and Surface Charge of the P(MAA-co-MBA) Microspheres
Microspheres of P(MAA-co-MBA) serve as substrates for the growth of the AgNPs, consequently, the size distribution, as well as the surface charge, have a close relationship with the nucleation and growth of the AgNPs. Figure 3 illustrates the results obtained from the DLS measurements of the polymerized P(MAA-co-MBA) microspheres dispersed in H2O prior to the growth of the AgNPs. Figure 3a reveals that the hydrodynamic radius of the microspheres in H2O is at 419 ± 109 nm, whereas the polydispersity index obtained by the correlation coefficient diagram (Figure 3b) is at 0.062, indicating monodisperse microspheres. Evidently, the resulting microspheres have a very narrow size distribution, which was further supported by SEM microscopy (Figure 4a), where the actual microsphere diameter is measured approximately at 200 ± 15 nm, whereas ζ-potential measurements affirm that the microspheres possess a strongly negative surface charge at −50 ± 5.34 mV, as illustrated in Figure 3c. Figure 4b presents the results of the EDS elemental analysis after the growth of the AgNPs. A broad excitation peak can be seen at 2.9 KeV which is attributed to metallic silver (15.22%,) whereas Ag2O or hybrid Ag/Ag2O are absent. According to the literature, these species are expected to exert smaller energies, around 0-1.35 KeV [46]. Carbon and oxygen peaks are attributed to the organic polymer counterpart, whereas gold was identified due to the sampling process for SEM analysis. Although elemental quantification of silver in EDS analysis was indicative, the results (15.22%) do not differ considerably when compared to the quantification analysis derived from TGA (23%); these differences could be attributed to the presence of Au in the EDS analysis.

Size Distribution and Surface Charge of the P(MAA-co-MBA) Microspheres
Microspheres of P(MAA-co-MBA) serve as substrates for the growth of the AgNPs, consequently, the size distribution, as well as the surface charge, have a close relationship with the nucleation and growth of the AgNPs. Figure 3 illustrates the results obtained from the DLS measurements of the polymerized P(MAA-co-MBA) microspheres dispersed in H 2 O prior to the growth of the AgNPs. Figure 3a reveals that the hydrodynamic radius of the microspheres in H 2 O is at 419 ± 109 nm, whereas the polydispersity index obtained by the correlation coefficient diagram (Figure 3b) is at 0.062, indicating monodisperse microspheres. Evidently, the resulting microspheres have a very narrow size distribution, which was further supported by SEM microscopy (Figure 4a), where the actual microsphere diameter is measured approximately at 200 ± 15 nm, whereas ζ-potential measurements affirm that the microspheres possess a strongly negative surface charge at −50 ± 5.34 mV, as illustrated in Figure 3c. Figure 4b presents the results of the EDS elemental analysis after the growth of the AgNPs. A broad excitation peak can be seen at 2.9 KeV which is attributed to metallic silver (15.22%,) whereas Ag 2 O or hybrid Ag/Ag 2 O are absent. According to the literature, these species are expected to exert smaller energies, around 0-1.35 KeV [46]. Carbon and oxygen peaks are attributed to the organic polymer counterpart, whereas gold was identified due to the sampling process for SEM analysis. Although elemental quantification of silver in EDS analysis was indicative, the results (15.22%) do not differ considerably when compared to the quantification analysis derived from TGA (23%); these differences could be attributed to the presence of Au in the EDS analysis.

Growth of AgNPs
It has been shown that increasing AgNO 3 concentration promotes both nucleation and growth of AgNPs [47]. In the current study, the Coulomb interaction between silver ions and the carboxyl-terminal groups of the polymer's chain was selected as the driving force for the initiation of nanoparticle growth instead of the initial AgNO 3 concentration. Therefore, during the stirring of the microspheres with AgNO 3 , aliquots were sampled every hour for 7 h, and a final sample was collected after 24 h of stirring. Each sample was centrifuged to remove nitrates and free silver ions, subsequently redispersed in the initial sample volume and finally reduced to AgNPs using 10 mM NaBH 4 . Each aliquot was measured in a UV-Vis spectrophotometer. Figure 5a depicts the UV spectra of the analyzed samples. The absorption peak shifted in different wavenumbers, yet a constant correlation between stirring time and peak shift was not observed. Table 1 shows the absorption band wavenumber, full width at half maximum (FWHM), and band area in correlation with stirring time. The FWHM, as well as the area of each band, were gradually increasing up to 5 h of stirring; from there on, a shift to the initial values was observed. This time effect can be observed in Figure 5b, where vials containing the corresponding aliquots after the Ag + reduction to AgNPs with NaBH 4 are illustrated. Specifically, after 24 h of stirring, the color tends to return to the initial shade acquired within the first hour of stirring.

Growth of AgNPs
It has been shown that increasing AgNO3 concentration promotes both nucleation and growth of AgNPs [47]. In the current study, the Coulomb interaction between silver ions and the carboxyl-terminal groups of the polymer's chain was selected as the driving

Growth of AgNPs
It has been shown that increasing AgNO3 concentration promotes both nucleation and growth of AgNPs [47]. In the current study, the Coulomb interaction between silver ions and the carboxyl-terminal groups of the polymer's chain was selected as the driving tion band wavenumber, full width at half maximum (FWHM), and band area in correlation with stirring time. The FWHM, as well as the area of each band, were gradually increasing up to 5 h of stirring; from there on, a shift to the initial values was observed. This time effect can be observed in Figure 5b, where vials containing the corresponding aliquots after the Ag + reduction to AgNPs with NaBH4 are illustrated. Specifically, after 24 h of stirring, the color tends to return to the initial shade acquired within the first hour of stirring.
(a) (b)  According to Hegde et al., AgNPs (<10 nm) with a spherical shape exhibit an absorption band with a maximum of 400 nm [48], whereas nanoparticle aggregation shifts the band to higher wavenumbers [49]. Furthermore, Garcia et al. showed the influence of the increased particle size distribution of AgNPs in the broadening of the absorption band. In particular, AgNPs with the same average size but wider size distributions exhibited broader FWHM in their absorption bands [50]. From the above results and observations, it is safe to deduce that the mechanism of electrostatic attachment of Ag + to the polymer microspheres is of a dynamic nature. Therefore, 3 h of P(MAA-co-MBA) stirring   According to Hegde et al., AgNPs (<10 nm) with a spherical shape exhibit an absorption band with a maximum of 400 nm [48], whereas nanoparticle aggregation shifts the band to higher wavenumbers [49]. Furthermore, Garcia et al. showed the influence of the increased particle size distribution of AgNPs in the broadening of the absorption band. In particular, AgNPs with the same average size but wider size distributions exhibited broader FWHM in their absorption bands [50]. From the above results and observations, it is safe to deduce that the mechanism of electrostatic attachment of Ag + to the polymer microspheres is of a dynamic nature. Therefore, 3 h of P(MAA-co-MBA) stirring microspheres in AgNO 3 solution was chosen as the optimum condition to proceed for further characterization, as well as antifungal activity assessment, since the absorption band is at 403 nm and the FWHM is at the average measured value, indicating the presence of spherical AgNPs with an average size distribution, respectively. Moreover, the FWHM to band area ratio (Table 1) shows that after 3 h of stirring, minimum particle aggregation is achieved.

Time of Stirring in AgNO 3 (h) Absorption Band Maximum (nm) Band FWHM Band Area
To correlate these results with the average size of the AgNPs, TEM analysis was conducted, and the results are presented in Figure 6. In Figure 6a (scale bar at 500 nm) large (~20 nm) spherical AgNPs are observed on the surface of the polymeric microspheres, whereas on the top right of the image a cluster of spherical nanoparticles can be seen, supporting the aggregation of AgNPs. At larger magnifications (Figure 6b-d), spherical AgNPs of a smaller diameter (<10 nm) are mostly observed, yet with large size distributions, thus further supporting the results obtained via the UV-Vis absorption spectra in terms of band area and band absorption maximum. Nevertheless, detached AgNPs can also be seen orbiting the P(MAA-co-MBA) surface in almost all images of Figure 6. This phenomenon is probably the result of the electron beam affecting the electrostatically attached AgNPs; a similar phenomenon was observed for gold nanoparticles according to Chen et al. in an attempt to manipulate gold nanoparticles utilizing an electron beam [51].
AgNPs of a smaller diameter (<10 nm) are mostly observed, yet with large size distributions, thus further supporting the results obtained via the UV-Vis absorption spectra in terms of band area and band absorption maximum. Nevertheless, detached AgNPs can also be seen orbiting the P(MAA-co-MBA) surface in almost all images of Figure 6. This phenomenon is probably the result of the electron beam affecting the electrostatically attached AgNPs; a similar phenomenon was observed for gold nanoparticles according to Chen et al. in an attempt to manipulate gold nanoparticles utilizing an electron beam [51].

Quantification of AgNPs
The mass percentage quantification of the AgNPs in the resulting composite nanostructure was accomplished via TGA. Figure 7a exhibits the TGA curve for the P(MAA-co-MBA) sample prior to the AgNPs growth (black curve), whereas the red curve represents the TGA curve after the growth of the nanoparticles (PMAA@Ag). The pristine PMAA-co-MBA has a residual mass of 11%, whereas the composite nanostructure exhibits a residual mass of 22%. Both curves start their thermal degradation at 318 °C and reach a plateau in terms of thermal degradation, at 450 °C and 423 °C for the PMAA-co-MBA and

Quantification of AgNPs
The mass percentage quantification of the AgNPs in the resulting composite nanostructure was accomplished via TGA. Figure 7a exhibits the TGA curve for the P(MAA-co-MBA) sample prior to the AgNPs growth (black curve), whereas the red curve represents the TGA curve after the growth of the nanoparticles (PMAA@Ag). The pristine PMAA-co-MBA has a residual mass of 11%, whereas the composite nanostructure exhibits a residual mass of 22%. Both curves start their thermal degradation at 318 • C and reach a plateau in terms of thermal degradation, at 450 • C and 423 • C for the PMAA-co-MBA and PMAA@Ag, respectively. According to Cárdenas et al. [52], the homopolymer PMAA exhibits a thermal degradation temperature at approximately 400 • C with minor deviations due to molecular weight differences. The 22% residual mass generated by the PMAA@Ag sample is the sum of the residual polymer (11% according to the black curve) and the residual Ag (11%). AgNPs are known to exhibit increased thermal conductivity; in our measurement, this can be observed by the rapid thermal degradation of the PMAA@Ag sample compared to the pristine PMAA sample. In addition, Hausner et al. [53], while studying the thermal decomposition of capped AgNPs observed that uncapped AgNPs of average size distribution of 6 nm start to sinter at 400 • C. In the present study, TEM analysis has confirmed the presence of AgNPs with sizing of 5-6 nm (Figure 6b-d). The two materials, PMAA-co-MBA and AgNPs, exhibit similar thermal degradation. As such, the mass percentage of the PMAA@Ag sample was subtracted from the mass percentage of the PMAA-co-MBA sample and was then plotted vs. temperature. These results are observed in Figure 7b. A similar method was applied by Kunc et al. for the quantification of silica amide functional groups utilizing TGA and NMR [54]. Indeed, at approximately 398 • C, a 12% mass increase can be observed for the PMAA@Ag sample which is attributed to the thermal degradation of smaller diameter (~6 nm) AgNPs. Therefore, the AgNPs percentage in the PMAA@Ag structure was estimated as 23% (11% residual and 12% thermally degraded at 398 • C).
of the PMAA-co-MBA sample and was then plotted vs. temperature. These results are observed in Figure 7b. A similar method was applied by Kunc et al. for the quantification of silica amide functional groups utilizing TGA and NMR [54]. Indeed, at approximately 398 °C, a 12% mass increase can be observed for the PMAA@Ag sample which is attributed to the thermal degradation of smaller diameter (~6 nm) AgNPs. Therefore, the AgNPs percentage in the PMAA@Ag structure was estimated as 23% (11% residual and 12% thermally degraded at 398 °C).

Antifungal Activity Assessment
For the antifungal activity evaluation, petri dishes were covered with a PDA layer loaded with increasing concentrations of PMAA@Ag nanostructures (0.5, 1, and 5 mg/mL) and incubated at their optimum growth conditions for up to ten days. As observed in Figure 8, after 48 h of incubation, fungal growth was clearly visible in the control samples, while no growth was observed in plates treated with PMAA@Ag at all concentrations examined. A. niger began to proliferate on the PMAA@Ag-treated agar on the 4th and 7th day of incubation, when the lower concentrations of 0.5 and 1 mg PMAA@Ag/mL, respectively, were employed. However, when 5 mg PMAA@Ag/mL solution, corresponding to approximately 1 mg AgNPs/mL, was used, the growth of A. niger was completely inhibited even after longer incubation times of up to ten days. Hence, PMAA@Ag nanostructures exhibit fungicidal activity at a concentration equal to 5 mg/mL, while at lower concentrations only fungistatic activity is observed.

Antifungal Activity Assessment
For the antifungal activity evaluation, petri dishes were covered with a PDA layer loaded with increasing concentrations of PMAA@Ag nanostructures (0.5, 1, and 5 mg/mL) and incubated at their optimum growth conditions for up to ten days. As observed in Figure 8, after 48 h of incubation, fungal growth was clearly visible in the control samples, while no growth was observed in plates treated with PMAA@Ag at all concentrations examined. A. niger began to proliferate on the PMAA@Ag-treated agar on the 4th and 7th day of incubation, when the lower concentrations of 0.5 and 1 mg PMAA@Ag/mL, respectively, were employed. However, when 5 mg PMAA@Ag/mL solution, corresponding to approximately 1 mg AgNPs/mL, was used, the growth of A. niger was completely inhibited even after longer incubation times of up to ten days. Hence, PMAA@Ag nanostructures exhibit fungicidal activity at a concentration equal to 5 mg/mL, while at lower concentrations only fungistatic activity is observed.
Although the biocidal effect and mode of action of AgNPs have long been known [55], their antifungal activity mechanism has only recently been elucidated. Candida albicans cells were employed for the clarification of the antifungal mode of action of AgNPs [56]. During exposure of C. albicans to AgNPs, significant changes to the fungal membranes were observed, resulting in the formation of pores, and, finally, cell death. The possible mechanism by which the AgNPs break down the membrane permeability barrier is by perturbing the membrane lipid bilayers, causing the leakage of ions and other materials, as well as forming pores and dissipating the electrical potential of the membrane.
AgNPs usually range in size from 1-200 nm, and as the particle size decreases, the surface area to volume ratio greatly increases. Consequently, the physicochemical, and especially the biological properties, of AgNPs are markedly improved as compared to the original material. Although AgNPs sizing from 1-10 nm are known to exert the highest antimicrobial activity [57,58] even at low concentrations, previous studies on fungi have shown that, in some cases, high concentrations of metallic NPs are needed for the antifungal activity to be exerted [4][5][6]59]. These interactions and their effect on membrane integrity are directly dependent on the size, the shape, and the AgNPs concentration. Apart from depending on the concentration of the AgNPs, the extent of inhibition also depends on the initial microbial population [60]. Specifically, Levard et al. [37] reported on these particles' interactions with intracellular substances from lysed cells, causing, therefore, their coagulation and the particles' exclusion from the liquid system. Although the biocidal effect and mode of action of AgNPs have long been known [55], their antifungal activity mechanism has only recently been elucidated. Candida albicans cells were employed for the clarification of the antifungal mode of action of AgNPs [56]. During exposure of C. albicans to AgNPs, significant changes to the fungal membranes were observed, resulting in the formation of pores, and, finally, cell death. The possible mechanism by which the AgNPs break down the membrane permeability barrier is by perturbing the membrane lipid bilayers, causing the leakage of ions and other materials, as well as forming pores and dissipating the electrical potential of the membrane.
AgNPs usually range in size from 1-200 nm, and as the particle size decreases, the surface area to volume ratio greatly increases. Consequently, the physicochemical, and especially the biological properties, of AgNPs are markedly improved as compared to the original material. Although AgNPs sizing from 1-10 nm are known to exert the highest antimicrobial activity [57,58] even at low concentrations, previous studies on fungi have shown that, in some cases, high concentrations of metallic NPs are needed for the antifungal activity to be exerted [4][5][6]59]. These interactions and their effect on membrane integrity are directly dependent on the size, the shape, and the AgNPs concentration. Apart from depending on the concentration of the AgNPs, the extent of inhibition also depends on the initial microbial population [60]. Specifically, Levard et al. [37] reported on these particles' interactions with intracellular substances from lysed cells, causing, therefore, their coagulation and the particles' exclusion from the liquid system.

Conclusions
Cross-linked poly (methacrylic acid) microspheres have been successfully prepared by utilizing the distillation precipitation method. Coulomb forces between silver cations and polymeric carboxyl side groups were employed as the driving force to promote the growth of silver nanoparticles on the surface of the P(MAA-co-MBA) microspheres,

Conclusions
Cross-linked poly (methacrylic acid) microspheres have been successfully prepared by utilizing the distillation precipitation method. Coulomb forces between silver cations and polymeric carboxyl side groups were employed as the driving force to promote the growth of silver nanoparticles on the surface of the P(MAA-co-MBA) microspheres, whereas UV-Vis and TEM microscopy revealed that these electrostatic interactions are of a dynamic nature. Our results evidenced that a 3 h stirring of P(MAA-co-MBA) microspheres in AgNO 3 was the appropriate time for the growth of AgNPs with an average size polydispersity. These results further suggest that the proposed methodology could result in nanostructures with tunable size and consequently, properties. The antifungal assessment against the growth of A. niger showed a fungistatic action in lower concentrations, whereas at higher concentrations the PMAA@Ag nanostructures exhibited fungicidal activity. The produced materials can be used for antifungal applications such as additives in filters, membranes, and storage packages in the medical, food, and cultural heritage (preventive conservation) sectors. However, it should be mentioned that, regarding food packages, AgNPs migration tests should be carried out; our future plans include the performance of these tests together with the evaluation of the antifungal activity of the obtained materials with respect to other fungi.