*3.4. Antibacterial Properties of PCL/Halloysite/ERY*

The antibacterial activity of HNT/ERY before and after the removal of residual ERY from HNT surfaces was assayed (Figure 6), and the obtained antibacterial properties of PCL/HNT and ERY are shown in Figure 7. Because PCL and HNT/PCL were not antibacterial active, as is clear from Figure 7, no inhibition zone was formed; the HNT/ERY and PCL/HNT/ERY nanofibers revealed significant antibacterial activity.

Zones of inhibition were observed around the HNT and ERY fibers and the = PCL/HNT and ERY fibers. After the successful incorporation of ERY into the carrier HNTs (proving that it was also a part of the prepared PCL/HNT fibers), the washing of the fibers in methanol followed by washing in water was conducted to ensure that the whole residual ERY of the surface was removed. Next, the antibacterial assay was repeated with washed materials. As is clearly shown in Figures 6 and 7, even though the zones of inhibition of washed samples were smaller, antibacterial activity was recorded again, which means that the antibiotic was incorporated into the HNT nanostructures. Accordingly, HNTs are an appropriate carrier for ERY.

Finally, the antibacterial activity was assayed when the PCL/HNT and ERY fibers were repeatedly (three times) replaced on fresh inoculated growth media. The results are shown in Table 2. These results support the idea of sustained release from the prepared fiber because the antibacterial effect was still observed after the second replacement (72 h) for *E. coli* and after the first replacement (48 h) for *S. aureus* (Table 2).

**Figure 7.** Comparison of the antibacterial activity of washed and unwashed PCL/HNT fibers with ERY assayed with the disk diffusion method on *E. coli* and *S. aureus.* The antibacterial activity was recorded as the inhibition zone diameter ± SE (standard error).

**Table 2.** Antibacterial activity of PCL/HNT and ERY fiber after 3x replacement.


#### *3.5. Mechanical Properties of PCL/HNT/ERY Nanofibers*

As it is clearly evident, the mechanical properties of investigated PCL nanofiber system were significantly influenced by the presence of both unmodified and HNT-loaded ERY (Figure 8, Table 3). The introduction of HNT particles into nanofibers significantly increased the Young's modulus and tensile strength. Furthermore, elongation at break remained unchanged for this system. The observed enhancement correlated well with the results obtained on PCL [40]. Stress transfer, volume replacement, and segmental immobilization [41–43] reinforcing mechanisms were responsible for this enhancement of mechanical performance. The loading of ERY into HNTs decreased Young's modulus to the value of the PCL while elongation at break was significantly increased. The observed trend re-affirmed the effect of plasticizers—small compatible molecules that are able to decrease the glass transition temperature of polymers—and suggested the good compatibility of ERY with the PCL/HNT nanofibers. More importantly, tensile strength also decreased upon ERY loading. However, the decrease was so slight that the values of the PCL/HNT and ERY systems were still more than 100% higher than those of the pure PCL system. A similar tensile strength enhancement (about 100%) was observed for PCL/gelatin/HNT microfiber system loaded with metronidazole [44].

**Figure 8.** Tensile strength, elongation at break, and Young's modulus of PCL, PCL/HNT, and PCL/HNT and ERY nanofibers.


**Table 3.** Young's modulus, tensile strength, and elongation at break of PCL, PCL/HNT, and PCL/HNT and ERY samples.

#### **4. Conclusions**

In this work, antibacterial, biodegradable, environmentally friendly, and nontoxic nanofibers with good biocompatibility and cost-effective production were prepared via the electrospinning of PCL and HNTs loaded with ERY. The PCL/HNT/ERY nanofibers exhibited outstanding antibacterial properties and resulted in the inhibition of both Gramnegative (*Escherichia coli*) and Gram-positive (*Staphylococcus aureus*) bacteria. We observed the gradual release of ERY out of the nanofibers, that was detected as repetitive antimicrobial activity after the material replacement on fresh prepared inoculated growth media. In addition to the antibacterial properties, the significant enhancement of mechanical properties was achieved via the incorporation of both unmodified and ERY-loaded HNTs, thus improving suitability of the system for medical applications. To summarize, PCL/HNT/ERY nanofibers with strong antibacterial effects have great potential as a progressive new biomedical material in both tissue engineering and a number of other biomedical applications.

**Author Contributions:** Conceptualization, V.K.; methodology, V.K.; investigation, V.K., M.K., K.G., F.O., P.O. and D.B.; writing—original draft preparation, V.K.; writing—review and editing, V.K., M.K, and Z.Š.; supervision, V.K.; funding acquisition, V.K and Z.Š. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors would like to thank the Slovak Grant Agency for financial assistance, project VEGA 1/0486/19 and VEGA 1/0411/22. Frantisek Ondreas greatly acknowledges the funding under the project LTAUSA19059 Inter-Excellence Grant from MEYS CR.

**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 authors also would like to thank I-Minerals Inc., Vancouver, BC, Canada, for providing of ULTRA HalloPure™ halloysite sample.

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