*2.6. Statistical Analysis*

The data are displayed as means ± standard deviations (SDs). To determine the statistical significance of the data, one-way analysis of variance (ANOVA) followed by Bonferroni's post-hoc test were performed. Values of \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001 were considered significant.

#### **3. Results and Discussion**

#### *3.1. Morphological Analysis*

In our previous studies, we showed the successful fabrication of fibrous materials obtained from polyesters by electrospinning [20,32,33]. In concrete, we found that for PLA with M<sup>W</sup> = 259,000 g/mol and MW/M<sup>n</sup> = 1.94, the optimal total polymer concentration for conducting electrospinning resulting in preparation of defect-free cylindrical fibers was 10 wt% in a DCM/EtOH solvent system [20]. However, it is necessary to study the effect of incorporation of a biologically active substance(s) on the morphologies of fibers, their physical–chemical properties, and their ability to inhibit the growth and penetration of pathogenic fungi.

SEM pictures of the obtained PLLA, PLLA/5-Cl8Q, and PLLA/K5N8Q fibers are presented in Figure 1. The electrospinning of PLLA resulted, reproducibly, in the fabrication of fibers with average fiber diameters of 1045 ± 320 nm (Figure 1a). The obtained diameters of the PLA fibers are in fairly good agreement with the literature data [34]. As can be easily seen using the selected conditions (concentration, solvent system, applied voltage, feeding rate, collector rotating speed, etc.), fibers with a cylindrical shape without defects and pores were obtained.

The addition of chemical fungicides (5-Cl8Q or K5N8Q) at a concentration of 10 wt% resulted in the preparation of stable solutions that did not alter the process of electrospinning and resulted in the fabrication of composite fibers with a cylindrical shape with a mean diameter close to that of the neat PLLA (Figure 1b,c). The average diameter of the fibers of the fibrous materials based on PLLA/5-Cl8Q and PLLA/K5N8Q was 1125 ± 300 nm and 1065 ± 250 nm, respectively. This is an indication that the addition of low molecular fungicides (derivatives of 8-hydroxyquinoline) did not lead to a significant change in the fiber morphology or diameters and distribution. These findings were confirmed by the measured values of the dynamic viscosities of the prepared solutions as well. The dynamic viscosity for PLLA, PLLA/5-Cl8Q, and PLLA/K5N8Q were relatively close and were 1350 cP, 1500 cP, and 1420 cP, respectively.

**Figure 1.** *Cont.*

**Figure 1.** Representative SEM images of the fibers of electrospun fibrous materials of PLLA (**a**), PLLA/5-Cl8Q (**b**), and PLLA/K5N8Q (**c**); magnification ×2500.

#### *3.2. Contact Angle Measurements*

It is well known that bacterial and fungal adhesion is influenced by the surface characteristics and the hydrophilic/hydrophobic balance of the host surface. For this reason, it is important to determine the values of the contact angle of the prepared electrospun materials that will contact the fungal species. The values of the water contact angles for all obtained samples were determined using distilled water droplets, and representative images of the droplets are shown in Figure 2. The PLLA fibrous material was hydrophobic, with a water contact angle of 117◦ ± 2.5◦ (Figure 2a). The measured value for the pure PLLA was close to the values found in the literature [35]. The measured contact angle values of the PLLA/5-Cl8Q and PLLA/K5N8Q composite fibrous materials were 120◦ ± 3 ◦ and 118.0◦ ± 2 ◦ , respectively (Figure 2b,c). The measured water contact angle values were close to those measured for the PLLA electrospun material. All of the obtained and studied electrospun fibers had water contact angle values ca. 120◦ and were hydrophobic.

**Figure 2.** Images of water droplets deposited on the surface of fibrous materials: (**a**) PLLA, (**b**) PLLA/5-Cl8Q, and (**c**) PLLA/K5N8Q.

#### *3.3. FTIR Spectroscopic Analysis*

FTIR spectroscopy was performed to characterize the prepared PLLA, PLLA/5-Cl8Q, and PLLA/K5N8Q fibrous materials, and the recorded spectra are shown in Figure 3. Characteristic bands for PLLA appeared at 1751 cm−<sup>1</sup> for the C=O groups and at 1182 cm−<sup>1</sup> for the C–O–C groups.

**Figure 3.** FTIR spectra of electrospun fibrous materials of PLLA, PLLA/5-Cl8Q, and PLLA/K5N8Q.

The characteristic stretching frequencies for C–O at 1080 cm−<sup>1</sup> and the bending frequencies for –CH<sup>3</sup> asymmetric and –CH<sup>3</sup> symmetric at 1452 cm−<sup>1</sup> and 1361 cm−<sup>1</sup> , respectively, were identified, also in accordance with the literature [36].

A new band appeared at 1500 cm−<sup>1</sup> , characteristic for the aromatic ring of the chemical fungicide in the PLLA/5-Cl8Q and PLLA/K5N8Q fibrous materials (Figure 3), in addition to the characteristic bands of PLLA [37], proving its presence in the electrospun composite materials.

Clearly, no molecular interaction between PLLA and the used fungicides was detected on the FT-IR spectra of the PLLA/5-Cl8Q and PLLA/K5N8Q composite fibrous materials.

#### *3.4. XRD Analysis*

Delivery systems based on nano- and microcarriers have been proven to be promising candidates for the delivery of poorly water-soluble or non-water-soluble compounds (drugs), wherein amorphization during thier encapsulation by the electrospinning process improves the dissolution of these compounds [12]. Therefore, it was of interest to study the changes in the crystallinity of chemical fungicides after their incorporation in composite electrospun fibrous materials. The crystallinity of the obtained fibers was determined by X-ray diffraction (XRD) analysis (Figure 4). The XRD pattern of PLLA and PLLA/5- Cl8Q materials and 5-Cl8Q powder, as well as of PLLA/K5N8Q fibrous material and K5N8Q powder, are presented in Figure 4a,b respectively. XRD patterns of 5-Cl8Q and K5N8Q (powder) with characteristic sharp diffraction peaks of the compounds were observed. These peaks showed that the fungicides (powders) were highly crystalline. The XRD spectra of the PLLA fibers showed a strong amorphous halo, proving that these materials have a typical amorphous structure. Moreover, in the spectra of the PLLA/5- Cl8Q and PLLA/K5N8Q composite materials, an amorphous halo was detected as well. This result indicates that each component in the composite fibrous materials prepared by electrospinning was in an amorphous state. This observation could be explained by the rapid drying of the jet during electrospinning, which impeded molecular motion. The obtained results are in accordance with the literature concerning the amorphization of poorly water-soluble drugs by electrospinning [38].

**Figure 4.** *Cont.*

**Figure 4.** X-ray diffraction pattern of (**a**) 5-Cl8Q powder and PLLA and PLLA/5-Cl8Q materials, and (**b**) K5N8Q powder and PLLA and PLLA/K5N8Q materials.

#### *3.5. Mechanical Properties*

Stress–strain curves of the PLLA, PLLA/5-Cl8Q, and PLLA/K5N8Q electrospun materials are presented in Figure 5. The PLLA material showed the highest tensile strength values. The obtained values are in good agreement with the literature data [20]. The determination of the mechanical characteristics of the PLLA/5-Cl8Q and PLLA/K5N8Q composite fibers showed that these materials possess similar mechanical properties, albeit a bit lower than those of the PLLA fibrous materials. This result indicates that the incorporation of 5-Cl8Q in fibrous membranes does not considerably change the mechanical characteristics of these membranes. The tensile strength of the PLLA/5-Cl8Q and PLLA/K5N8Q composite fibrous materials was ca. 2.5 MPa, while the tensile strength of the PLLA fibrous materials reached 3.4 MPa. The slight decrease in mechanical characteristics might be due to the incorporation of low molecular weight chemical fungicides in the PLA matrix, which might have generated weak spots when the tensile test was carried out.

**Figure 5.** Stress–strain curves of electrospun materials: (1) PLLA, (2) PLLA/5-Cl8Q, and (3) PLLA/K5N8Q.

#### *3.6. Cumulative Drug Release Analysis*

The electrospinning method is often used for encapsulation of drugs for delivery. There are many data in the literature concerning the release rates of incorporated drugs in different polymer matrixes showing diverse behavior: Some show an initial burst release of the drug, while others show a more controlled release of the drug over a longer duration. This is due to the fact that many parameters could influence drug release, e.g., the molecular characteristics of the polymer, the polymer crystallinity and hydrophobicity, the nature of the drug and its crystallinity, the compatibility of the drug with the polymers matrix, the fiber morphology and diameters, the presence of defects along the fibers. Therefore, the same drug loaded in different polymer matrixes or different drugs incorporated in same polymer matrix may exhibit different release profiles.

The release of 5-Cl8Q and K5N8Q from PLLA fibrous matrixes was studied, and their release profiles are shown in Figure 6. Initially, both drugs showed a relatively burst release from the PLLA fibrous matrix. However, K5N8Q was released in a higher amount compared to 5-Cl8Q for the same duration. For instance, the released K5N8Q was 10.5% and 21.4% for 30 and 60 min. For the same time durations, the released 5-Cl8Q was 6.7% and 9.3%, respectively. This difference in the release profiles could be due to the different natures of the drugs and their water solubility. K5N8Q is a partially water-soluble drug favoring a more rapid release. On the contrary, 5-Cl8Q is water-insoluble, which hampers its release. After 50 h, the amounts of the released 5-Cl8Q and K5N8Q were 52.8% and 72.5%, respectively.

**Figure 6.** Release profiles of 5-Cl8Q and K5N8Q from PLLA fibers: PLLA/5-Cl8Q (♦) and PLLA/K5N8Q (). The results are presented as the average values from three separate measurements with the respective standard deviation; acetate buffer/lactic acid (96/4 *v*/*v*), pH 3, 37 ◦C, ionic strength 0.1.

#### *3.7. Antifungal Activity of the Fibrous Membranes*

The two main fungi causing esca disease are *P. chlamydospora* and *P. aleophilum*. The wounds on vines caused during pruning are the main point of entry for the penetration of fungal spores in grapevines. Because there is no direct way to fight esca, there is a rising demand for the development of novel plant protective agents and materials that are non-toxic but are efficient against esca.

Although there are some data with respect to the antifungal activity of 8-hydroxyquilonine derivatives against *Candida* species, there are no data concerning their effects on *P. chlamydospora* and *P. aleophilum*, which are the main causative agent of esca disease. In our

previous study, we determined the minimum inhibitory concentration (MIC) of 5-Cl8Q against *P. chlamydospora* and *P. aleophilum*, and it was found to be 0.75 µg/mL for both strains [24]. The MIC determined by us for K5N8Q was 12.5 µg/mL and 25 µg/mL for *P. chlamydospora* and *P. aleophilum*, respectively.

The antifungal activity of the electrospun fibrous materials (diameter 17 mm) was determined by carrying out antifungal tests against *P. chlamydospora* and *P. aleophilum*.

Figure 7 presents the observed zones of inhibition after contact of the fibrous materials with the fungal cells. The loading of 5-Cl8Q in the composite fibrous materials that were laid in contact with *P. chlamydospora* resulted in complete inhibition of fungal growth. Moreover, there was a wide inhibition zone around the PLLA/5-Cl8Q disc put in contact with *P. aleophilum* (4.7 cm). Additionally, the incorporation of K5N8Q resulted in wide zones of inhibition as well. The diameters of the inhibition zones around the PLLA/K5N8Q discs were 6.2 cm and 4.0 cm against *P. chlamydospora* and *P. aleophilum*, respectively. From the obtained results, it is easily seen that *P. chlamydospora* is more vulnerable to treatment with the used 8-hydroxyquinoline derivatives.

**Figure 7.** Digital pictures of the zones of inhibition against *P. chlamydospora* and *P. aleophilum* after contact of the fibrous materials with fungi cells. The material type is indicated at the top of each column. The cell type is marked in the left of each row.

> The results obtained in the present study demonstrate that composite fibrous materials containing hydroxyquinoline derivatives have strong antifungal activity. In contrast, neat PLLA fibrous materials do not change the fungal growth or exhibit any antifungal activity.

> In the present study, the barrier efficacy of PLLA, PLLA/5-Cl8Q, and PLLA/K5N8Q electrospun fibrous materials were studied as well. For this purpose, 20 mL of conidia suspension was passed through each fibrous material (diameter 45 mm) using a filtration device. Initially, we determined the size of the *P. chlamydospora* and *P. aleophilum* conidia in the fungal suspension using SEM analysis. Figure 8 presents the used conidia.

**Figure 8.** SEM micrographs of the (**a**) *P. chlamydospora* and (**b**) *P. aleophilum* conidia.

The diameter and length of the *P. chlamydospora* conidia were ~0.75–1.2 µm and 1.8–2.3 µm, respectively. The measured diameter and length of the *P. aleophilum* conidia were ~1.1–1.5 µm and 2.5–3.5 µm, respectively. The initial concentration in the filtration experiments was 1 <sup>×</sup> <sup>10</sup><sup>7</sup> conidia/mL for both strains. After passing through the electrospun discs, the determined spore concentration was 1.6 <sup>×</sup> <sup>10</sup><sup>3</sup> , 1.3 <sup>×</sup> <sup>10</sup><sup>3</sup> , and 1.4 <sup>×</sup> <sup>10</sup><sup>3</sup> for the PLLA, PLLA/5-Cl8Q, and PLLA/K5N8Q materials, respectively. This result reveals that the final conidia concentration decreased significantly. However, some conidia passed through all of the fibrous materials in this study.

It was of interest to determine not only the ability of the materials to impede the penetration of fungal spores, but also to study if the fibrous materials impede the growth of pathogenic fungi remaining in the material after filtration. Therefore, after filtration, we placed the used disks on a surface of solid agar in a Petri dish in order to determine the growth of the remaining fungi in the fibrous discs. The Petri dishes were incubated for 96 h at 28 ◦C, and then the fungal growth was determined. Figure 9 presents the growth of *P. chlamydospora* on the fibrous materials' surface. It was found that the PLLA fibrous material used in the filtration experiments developed colonies of *P. chlamydospora* (Figure 9a). The developed colonies showed that this material did not possess antifungal activity. PLLA/5- Cl8Q and PLLA/K5N8Q, which were placed in suitable conditions for the development of remaining spores in the materials, impeded the fungal growth, resulting in compete fungal inhibition (Figure 9b,c).This result indicates that the fungi remaining in the PLLA/5-Cl8Q and PLLA/K5N8Q materials after filtration could not grow due to the antifungal activity of the obtained composite materials.

**Figure 9.** Digital images of the growth *P. chlamydospora* on the fibrous materials after spore filtration: (**a**) PLLA, (**b**) PLLA/5- Cl8Q, and (**c**) PLLA/K5N8Q.

### **4. Conclusions**

Novel micro- and nanofibrous materials of PLLA, PLLA/5-Cl8Q, and PLLA/K5N8Q were successfully electrospun. The obtained composite materials were hydrophobic with good mechanical properties. The incorporation of 5-Cl8Q or K5N8Q into the PLLA fibers imparted to them a considerable antifungal activity against *P. chlamydospora* and *P. aleophilum*. These features demonstrate that the obtained composite fibrous materials could be a potential candidate for application in agriculture for grapevine protection against esca-associated fungi.

**Author Contributions:** M.S., I.R., and N.M. conceived the original concept. N.N. and M.S. conducted the experiments and characterized the electrospun fibrous materials. P.T. performed the drug release analysis. M.N. performed the microbiological assessments of the obtained materials. M.S., N.M., and I.R. wrote the manuscript. M.S., N.M., I.R., and M.N. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Bulgarian National Science Fund, grant number KP-06- OPR03/2.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The financial support from the Bulgarian National Science Fund (grant KP-06- OPR03/2) is gratefully acknowledged.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

