**3. Discussion**

The current study evaluated the antifungal activity of TC-NCs and OV-NCs against selected fungal strains that contaminate different natural matrices, indoor air and materials (Table 3). The results evidenced that both TC-NCs and OV-NCs showed in vitro antifungal activity against all tested isolates with an MIC range of 0.125–0.25 mg/mL (Table 4). This activity is two to four times greater than that of pure oils. These results highlight the effectiveness of EO encapsulation in a nanometric structure. In fact, the nanocapsules were able to protect essential oils from degradation phenomena and increase the transport and diffusion mechanisms of essential oils through the cell membrane [30].

Moreover, the same MIC and MFC values were observed for both nanocapsule suspensions in respect to the different strains, with the one exception of the *Bjerkandera adusta* strain, against which the OV-NCs showed more activity than the TC-NCs. The results were imputable to the chemical compositions of commercial essential oils. Although the main bioactive oxygenated monoterpenes (thymol and carvacrol) were present in different quantities in the two essential oils, the sum of their relative percentages was of the same order of magnitude (Table 1). Generally, the growth-inhibiting effects were attributable to the major components, thymol and carvacrol, but the bioactive monoterpene hydrocarbons, *p*-cymene and γ-terpinene present in both essential oils in non-negligible quantities could have given a valuable contribution to the antifungal properties [31].

Regarding the physicochemical characterization of the nanocapsules, the TC-NCs and OV-NCs showed nanodimensions and good PDIs indicating the presence of monodisperse species. This characteristic is crucial for achieving optimal biological activity results [32]. Acidic residues on the PCL polymer and the presence of tween 80 (a neutral surfactant covering the nanoparticle wall) were in agreemen<sup>t</sup> with the observed low and negative values of the zeta potential. Moreover, these values were very similar to those previously reported by us [28,29] and by other authors [33,34] for stable poly(ε-caprolactone) nanocapsules.

The good encapsulation in the range of 80–84% and the loading capacity of 51–52% highlighted the ability of the PCL to effectively encapsulate essential oils. Furthermore, the ease of preparation of these nanosystems and their stability in aqueous environments could make them useful in different application fields.

PCL is a biocompatible and biodegradable polymer approved by the Food and Drug Administration for drug delivery application. It is a polymer with a low cost and high hydrophobicity and stability. Moreover, PCL is characterized by slow degradation, which makes it potentially more suitable than other polymers, such as polyglycolide (PGA) and poly D, L-lactide (PDLA), when a slow release of the bioactive is required [35].

We have reported here the first example of PCL-based nanocapsules containing essential oils with more enhanced antifungal activity than pure essential oils against a panel of fourteen fungal strains. In the literature, the antifungal activity of essential oils encapsulated in different nanocarriers was also reported. There are many examples of encapsulated EOs in chitosan nanostructures with antifungal activity [36], the majority of which were obtained by the emulsion ionic gelation technique. In a recent work, Hasheminejad et al. [37] demonstrated that clove essential oil encapsulated by chitosan nanoparticles inhibited the growth of *Aspergillus niger*, isolated from spoiled pomegranate, at a concentration of 1.5 mg/mL. Instead, against *Aspergillus flavus*, Khalili et al. [38] reported an MIC value of 300 mg/L by using a chitosan benzoic acid nanogel containing thyme essential oil under sealed conditions. *Fusarium graminearum* was inhibited by a chitosan-encapsulating *Cymbopogon martini* essential oil [39], with an MIC value of around 0.42 mg/mL.

Nanoemulsions of oregano EO, prepared by Bedoia-Serna et al. [40], showed antifungal effects against *Cladosporium* sp., *Fusarium* sp. and *Penicillium* sp. The authors found that for *Fusarium* sp., unlike with *Penicillium* sp., the encapsulation of the oregano essential oil enhanced its antifungal effect.

β-Cyclodextrin was also used to entrap essential oils with antifungal activity through the formation of an inclusion complex. Capsules of β-cyclodextrin containing clove and Mexican oregano EO were active against *Fusarium oxysporum* [41]. An inclusion complex based on β-cyclodextrin and *Litsea cubeba* EO presented effective antifungal activity against three main fungi in citrus, including *Penicillium digitatum*, *Penicillium italicum* and *Geotrichum citri-aurantii* [42].

The antifungal activity of inorganic nanoparticles loaded with essential oils was reported by Weisany et al. In particular, the authors showed that encapsulation of *Thymus daenensis* and *Anethum graveolens* in silver nanoparticles enhanced their fungicidal activity against the plant pathogen *Colletotrichum nymphaeae* [43]. The same authors proved that copper nanoparticles containing thyme and dill EOs were able to reduce the mycelium growth and strongly inhibit the germination of conidia of *Colletotrichum nymphaeae* [44].

Unexpectedly, in the literature, no example of nanostructured systems containing essential oils effective against the fungi *Mucor circinelloides*, *Exophiala xenobiotica*, *Purpureocillium lilacinum*, *Pleurotus eryngii*, *Bjerkandera adusta* or *Phanerochaete chrysosporium* has been reported.

Although the antibacterial activity of EO nanocapsules based on PCL have been evaluated [28,45,46], less interest has been paid to their antifungal activity. The co-encapsulation of chloramphenicol with lemongrass essential oil in PCL-Pluronic composite nanocapsules was reported by Wang et al. [47]. This nanosystem showed considerably enhanced activity against methicillin-resistant *Staphylococcus aureus* and *Candida* sp. Tea tree essential oil loaded in PCL nanocapsules exhibited antifungal activity against *Trichophyton rubrum*, an etiological agen<sup>t</sup> of superficial human mycosis [48].

In light of this, our results could provide a valuable contribution to the use of encapsulated essential oils as effective natural fungicides, as an alternative to the synthetic ones responsible for increasing resistant fungal strains.

#### **4. Materials and Methods**

All solvents were of analytic grade. In order to prepare the essential oil-loaded nanocapsules, sorbitan monostearate (SM) and poly(ε-caprolactone) (PCL) (Mn 45000) were obtained from Sigma-Aldrich (Milan, Italy) and polysorbate 80 (Tween 80) was obtained from Fisher Chemical (Fisher Scientific, Geel, Belgium). Water Chromasolv Plus for HPLC (Honeywell Riedel-de-Haën, Seelze, Germany) was used. Commercial essential oils of oregano and thyme were provided by Esperis S.p.A., (Milan, Italy) and by Flora s.r.l. (Lorenzana, Pisa, Italy), respectively. A standard mix of *n*-alkanes C9–C22 was purchased by Alltech (Italy).

#### *4.1. Characterization of Essential Oils by Gas Chromatography–Flame Ionization Detection (GC–FID) and Gas Chromatography–Mass Spectrometry (GC–MS)*

GC–FID and GC–MS analysis were performed on a Shimadzu GC-17A and a Shimadzu GCMS-QP5050A, respectively. For both analyses, the same fused silica capillary column (Supelco SPBTM-5 15 m, 0.1 mm, 0.1 mm) was used.

The operating conditions for both runs were as follows: injector temperature 250 ◦C; detector temperature 280 ◦C; carrier gas helium (1 mL/min); split mode (1:200); and volume of injection 1 mL (4% essential oil/CH2Cl2 *v*/*v*). The heating ramp was as follows: 60 ◦C for 1 min, 60–280 ◦C at 10 ◦C/min, then 280 ◦C for 1 min. The relative percentages of compounds in each essential oil were determined from the peak areas in the GC–FID profiles. The mass spectrometer operating conditions were as follows: ionization at 70 eV and an ion source temperature of 180 ◦C. Mass spectral data were acquired in the scan mode in the m/z range of 40–400. Oil solutions were injected with the split mode (1:96) [49].

The identity of components was determined, comparing their retention indices relative to the C9–C22 *n*-alkanes on the SPB-5 column, computer matching of spectral MS data with those from National Institute of Standard and Technology (NIST) Mass Spectral Library (MS) 107 and 21 [50] and the comparison of the fragmentation patterns with those reported in the literature [51].

#### *4.2. Preparation of Essential Oil-Loaded Nanocapsules (EO-NCs)*

The EO-loaded nanocapsules were prepared according to the preparation described in Granata et al. [28], but with slight changes, above all concerning scaling up by about three times. A solution of sorbitan monostearate (112 mg), PCL (320 mg), and EO (1.0 g) in acetone (80 mL) at 30 ◦C was poured into an aqueous solution of polysorbate 80 (275 mg in 160 mL of pure water) while stirring. The suspension was stirred for another 10 min at 25 ◦C. Then, the organic solvent was removed carefully under vacuum conditions (bath at 30 ◦C, pressure gradually reduced from 550 to 100 mbar in 1 h, and then reduced at 90 mBar and kept constant for 10 min). Finally, in order to complete the solvent evaporation, the mixture was fluxed by N2 (1 h, atmospheric pressure), obtaining the EO-NC suspension (160 mL). The empty NC suspension was achieved under the same conditions but without essential oil.

#### *4.3. Physicochemical Characterization of EO-NCs*

4.3.1. Encapsulation Efficiency (EE) and Loading Capacity (LC) of EO-NCs

The total amount of essential oil in the EO-NC suspensions was determined by UV −vis spectroscopy (8453 UV-Visible Spectrophotometer, Agilent Technologies, Milan, Italy) over wavelengths ranging from 250 to 450 nm (λmax 274). A 20 μL EO-NC suspension was diluted with 2 mL of acetonitrile, and the absorbance at 274 nm was registered and corrected from the small absorbance due to the other components. The OV or TC concentration was determined from the value of the correct absorbance compared to the linear calibration curve of respective EO, obtained by plotting the absorbance at 274 nm of twelve solutions containing different concentrations of OV (from 11 to 118 μg/mL, R<sup>2</sup> = 0.9999) or eleven solutions containing TC (from 10 to 102 μg/mL, R<sup>2</sup> = 0.9999). The ultrafiltration centrifugation technique (Nanosep 30K Omega, Pall Life Science, Milan, Italy; 90 min at 3500× *g*) was used to estimate the free essential oil. The suspension (500 μL) of the EO-NC was ultrafiltrated, and then an aliquot (60 μL) of the filtrate was diluted with 2 mL of acetonitrile. As described above, the free essential oil amount was determined by the absorbance at 274 nm of the resulting solution. From the total and free amounts of essential oil, the encapsulation efficiency was calculated:

$$\text{EE (\%)}=\text{([EO]}\_{\text{tot}}-[\text{EO]}\_{\text{free}})/[\text{EO}]\_{\text{tot}}\times 100\tag{1}$$

where [EO]tot and [EO]free are the total and free amounts of essential oil in the EO-NC suspensions, respectively.

The loading capacity was calculated by the following equation:

$$\text{LCC} \left( \% \right) = \left( \text{mass of loaded EO} \right) / \left( \text{mass of loaded nanoribquessules} \right) \times 100 \tag{2}$$

4.3.2. Particle Size, Polydispersity and Zeta Potential Measurements

The mean diameter (z-average), the polydispersity index (PDI) and the I-weighted distribution of the EO-NCs were obtained at 25 ◦C by dynamic light scattering (DLS). The zeta potential (*ζ*) values were determined by electrophoretic light scattering (ELS). DLS and ELS experiments were performed on a Zetasizer Nano ZS-90 (Malvern Instruments, Cambridge, UK), and data were analyzed using Zetasizer Version 7.02 software. For these purposes, the EO-NC suspensions were previously diluted (1:200, *v*/*v*) with pure water or with a pre-filtered (0.45 μm) 10 mM NaCl aqueous solution to carry out DLS or ELS, respectively.

Each experiment was replicated at least twice, and measurements performed at least three times. All data are expressed as mean ± standard deviation (SD).

#### *4.4. Microorganisms and Growth Conditions*

The fungal strains used in this study, their environment of isolation and sources are detailed described in Table 3 (*Aspergillus fumigatus*, *Aspergillus flavus*, *Penicillium rubens*, *Penicillium citrinum*, *Cladosporium aggregatocicatricatum*, *Cladosporium herbarum*, *Fusarium*

*oxysporum*, *Geotrichum candidum*, *Mucor circinelloides*, *Exophiala xenobiotica*, *Purpureocillium lilacinum*, *Pleurotus eryngii* CCBAS471, *Bjerkandera adusta* CCBAS232 and *Phanerochaete chrysosporium* CCBAS570). The fungal strains were grown at 26 ◦C on malt extract agar (MEA) for 5 days.

#### *4.5. Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC)*

The MIC and MFC values of the nanoencapsulated essential oils (EO-NCs) and pure EOs were evaluated with a panel of fourteen fungal strains (Table 3). Fungal suspensions were prepared according to de Lira Mota et al. [52] by washing the surface of the MEA slant culture with 5 mL of sterile saline and shaking the suspensions for 5 min. The resulting mixture of sporangiospores and hyphal fragments was withdrawn and transferred to a sterile tube. After heavy particles were allowed to settle for 3–5 min, the upper suspension was collected and vortexed for 15 s. Fungal suspensions were adjusted to a final concentration of 10<sup>6</sup> conidia mL−<sup>1</sup> in malt extract broth (MEB). Nine milliliters (9 mL) of MEB containing one hundred microliters of the fungal suspension was distributed into incubation flasks. Encapsulated essential oils of oregano (OV-NC) and thyme (TC-NC) at different concentrations (0.05 to 0.5 mg/mL) and pure oregano (OV-EO) and thyme (TC-EO) at concentrations ranging from 0.05 to 5 mg/mL were added individually to the flasks. The flasks were then incubated at 26 ◦C for 7 days. After incubation, those fungal strains that did not show any growth were transferred to fresh MEA plates without EO-NCs for an additional 7 days at 26 ◦C to confirm which concentration had a fungicidal effect. The concentration unfavorable for growth revival during the transfer experiment was taken as the MFC, and this effect was identified as fungicidal. The lowest concentration of EOs or EO-NCs that prevented visible fungal growth and allowed a revival of fungal growth during the transfer experiment was considered the MIC.
