**1. Introduction**

Halloysite nanotubes (HNTs) are natural, nontoxic, biocompatible, eco-friendly, and low-cost materials recognized by the Environmental Protection Agency as nanomaterials (EPA 4). Presently, HNTs play a significant role in drug-carrier systems suitable for different biomedical applications, e.g., tissue engineering [1,2].

HNTs, naturally occurring in 1:1 layered aluminosilicate clay, consist of aluminum and silicon oxide layers rolled into tubes. The layers are rolled into tubes because of differences in the sizes of silicon and aluminum ions [3]. The typical length of the nanotubes is about 1–2 μm. Their outer and inner diameters range from 50 to 100 nm and from 10 to 50 nm, respectively [4,5]. For biomedical applications, the most significant advantages of HNTs

**Citation:** Khunová, V.; Kováˇcová, M.; Olejniková, P.; Ondreáš, F.; Špitalský, Z.; Ghosal, K.; Berkeš, D. Antibacterial Electrospun Polycaprolactone Nanofibers Reinforced by Halloysite Nanotubes for Tissue Engineering. *Polymers* **2022**, *14*, 746. https://doi.org/10.3390/ polym14040746

Academic Editors: Ariana Hudita and Bianca Galˇ a¸ˇteanu

Received: 20 January 2022 Accepted: 8 February 2022 Published: 15 February 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

compared to other tubular silicates are that they present a unique combination of structure, natural availability, rich functionality, good biocompatibility, and cytotoxicity [2,3,6–9].

One of the most remarkable features of HNTs is their different surface chemistries at the inner and outer sides of the tubes: silica sheets make up the external surfaces of the tubes and aluminum oxide makes up the inner (lumen) surface chemistry. Furthermore, alumina has a positive charge up to pH 8.5, and silica has a negative charge at pH values above 1.5 [10,11]. Due to differently charged outer and inner sides, it is possible to utilize HNTs as multifunctional nanocontainers for the selective modification of the outer and inner sides of nanotubes [7,11–16].

Halloysite's inner diameter fits well to macromolecules and proteins [6]. In this regard, drugs of smaller molecular size are typically vacuum-loaded within the inner lumen of the nanotube, and drugs with larger molecular size can attach to the outer surface of the halloysite [17].

HNTs have also been successfully used as low-cost nanocontainers for several antibiotics such aminoglycoside gentamicin [18] and β-lactam antibiotic amoxicillin [19]. In addition, a wide range of applications of vancomycin-loaded halloysite nanotubes have been presented in alginate-based wound dressing [20] and silk fibroin hydrogel applicable for bone tissue engineering [21].

Electrospinning is a technology for the fabrication of continuous nanofibers with a simple setup [22]. Electrospun nanofibers can be prepared from natural or synthetic polymers or their blends. In the past decade, significant progress has been achieved in researching advanced electrospun nanofibers for biomedical applications or one-dimensional nanofibers made of intrinsically conducting polymers [23]. Recent advances in the electrospinning of functional scaffolds for tissue engineering and nanofiber scaffolds were summarized in the work of Hanumantharao et al. [24]. Simultaneously with the improvement of electrospun process technology, a lot of effort is being made to study new types of antimicrobial nanoparticles or make the known ones much more effective against microbial effects [25–28].

At present, there have been many examples where a biodegradable polymer matrix was combined with antimicrobial nanofillers (e.g., metals and/or metal oxides) for the preparation of polymer nanocomposites for biomedical applications [29–31]. An extensively used synthetic, biodegradable, semi-crystalline polymer used for biomedical applications is polycaprolactone (PCL) [32]. PCL is well known for its versatile use, biocompatibility, chemical stability, thermal stability, slow biodegradation (around 24 months), tissue compatibility, and easy processing [31,33]. PCL is often used in biomedicine as an FDA-approved material in the form of nanofibers that have evolved as controllable drug delivery systems [34,35]. Furthermore, due to its slow degradation rate, PCL is a preferred polymer mainly used as a long-term drug delivery carrier. Another advantage of PCL is its compatibility with a wide range of drugs, which provides a homogenous distribution of predominantly lipophilic drugs in the carrier matrix due to its hydrophobic nature [36]. Moreover, in our earlier work, it was confirmed that except for an important and strong reinforcing effect, all studied PCL/Gel nanofibers with an HNT content from 0.5 to 9.0 wt.% were non-toxic and had no effect on cell behavior [35]. Thus, through a combination of lowstrength PCL with drug-loaded halloysite, it is possible prepare reinforced biodegradable polymer nanocomposites with regular drug release.

In this work, electrospun nanofibers based on PCL and drug-loaded HNTs were studied. HNTs were used as a nanocarrier for erythromycin (ERY) as a model antibacterial active compound with wide range of antibacterial activity on both Gram-positive and Gram-negative bacteria. ERY—a macrolide antibiotic used to treat a number of bacterial infections, e.g., respiratory tract infections and pelvic inflammatory disease—was used for HNT loading. In addition, erythromycin is clinically used in dermatology as a very effective topical antibiotic drug in treating bacterial skin disease.

ERY is soluble in methanol but has minimal solubility in an aqueous solution and presents acid instability, which limit its broader application. Therefore, drug-carrier systems based on HNTs and ERY have great potential to overcome these weaknesses. Moreover, cheap ERY was chosen as a model substrate for another type of pH-sensitive macrolide antibiotics intensively studied by our group [37].

#### **2. Materials and Methods**

#### *2.1. Materials*

PCL CAPA® 6800 (Mw = 80,000) and Erythromycin E6376 were obtained from Sigma-Aldrich Saint Louis, MO 63103, USA and ULTRA HalloPure, respectively, produced by I-Minerals Inc., Vancouver, BC, Canada. The ULTRA HalloPure comprised purified HNTs with 93.5% of halloysite, 6.1% of kaolinite, and 0.4% of quartz from Dragon Mine in Utah of USA, produced by Applied Minerals Inc., New York, NY, USA. The dimensions are presented in Table 1. Methanol and distilled water were purchased from Lachner s.r.o., Neratovice, Czech Republic.

**Table 1.** Structural parameters of ULTRA HalloPure.


#### *2.2. Loading of a Drug in Halloysite Nanotubes*

The loading of active agents to halloysite is based on the diffusion of molecules from an external solution into the inner part of HNTs due to the concentration gradient. The evaporation of the solvent under the vacuum elevates the concentration of the active agents in the solution and enhances the diffusion rate. Therefore, fast-drying solvents with low viscosities such as acetone or ethanol are preferable for organic substances [38]. To eliminate potential water on the outer part of HNTs before the loading procedure, HNTs were dried in the oven for 2 h at 150 ◦C. Since ERY is soluble in methanol, after the drying, it was dissolved in methanol. HNTs were dispersed in the ERY solution and sonicated for 20 min. The rate of halloysite to erythromycin was 60:40. After the sonification, halloysite loaded with ERY was dried in a vacuum and washed with distilled water using a 0.2 μm membrane filter. Residual unloaded ERY was removed with methanol (4 h).

HNTs were dried in the oven for 2 h at 150 ◦C. We dissolved 400 mg of the drug in 25 mL of methanol. We dispersed 600 mg of dried halloysite in a drug solution (ratio 40:60 ERY:HNT), which was then sonicated for 20 min. Then, this dispersion was dried in a vacuum. After vacuum drying, it was washed with distilled water using a 0.2 μm membrane filter. Residual unloaded ERY was removed with methanol (4 h).

#### *2.3. Optimization of Electrospinning Process Parameters*

The electrospinning process for PCL composites (Figure 1) was optimized and previously described by our group [29]. Briefly, a solution of 10% w/v PCL was prepared in a mixture of chloroform and methanol in a ratio of 4:1 and continuously stirred for 2 h. Then, the mixture was kept unstirred for another 15 min to remove any bubbles present in the solution. Prepared HNTs loaded with ERY were added to the PCL solution and stirred for another 1 h. Electrospun fibers were fabricated using a Spellman high voltage power source (Spellman High Voltage Electronics Corporation, New York, NY, USA) and syringe pump (New Era Pump Systems, Inc., New York, NY, USA). The electrospinning apparatus consisted of a 10 mL syringe that was integrated with a grounded electrode, and the needle diameter was 0.41 mm. The distance between tee collector and source was kept to 13 cm. One thin aluminum sheet was fixed over the static collector. The feeding rate for the electrospinning solution was set to 1 mL/h at a voltage of 25 kV. The temperature for the location varied between 21 and 29 ◦C, and the humidity varied from 73% to 93%.

**Figure 1.** Scheme of electrospun nanofiber preparation.

#### *2.4. Scanning Electron Microscopy*

For the needs of scanning electron microscopy (SEM), the sample's surface was covered in gold with a Sample Preparation System Quorum Technologies Q150R S/E/ES sputter coater evaporator (Quorum Technologies, Laughton, England, and the micrographs were obtained with an FIB Microscope Quanta 3D 200i (FEI Company, Tokyo, Japan) in a secondary electron mode at different magnitudes. The surface morphologies of pure and modified fibers were obtained.

### *2.5. Transmission Electron Microscopy*

The structure of HNTs loaded with ERY was characterized by transmission electron microscopy (Jeol TEM 1200EX, JEOL Ltd., Tokyo, Japan) at an accelerating voltage of 100 kV. The sample was dispersed on a copper grid with carbon support film.

#### *2.6. Antibacterial Activity*

The antibacterial activity of HNT/ERY before and after removing residual ERY from HNTs was assayed. The antibacterial activity of PCL/HNT/ERY nanofibers was assessed with the diffusion method by placing the HNT, HNT:ERY, 1 cm<sup>2</sup> of prepared PCL fiber, PCL/HNT fiber, and PCL/HNT and ERY fiber on the inoculated (106 cells/mL) MHA (Mueller Hinton Agar) growth media. For inoculation, the model bacteria *S. aureus* CCM 3953 (Czech Collection of Microorganisms) and *E. coli* CCM 3988 were used. The antibacterial activity of prepared nanofibers was evaluated as the occurrence of a sterile zone of inhibition around the modified HNTs with ERY and modified fibers of PCL/HNT and ERY, respectively, after cultivation for 24 h at 37 ◦C. With the aim to wash out the residual ERY from the surface of the prepared materials, modified HNTs with ERY and the PCL/HNT and ERY fibers were washed two times in methanol and two times in water. The antibacterial activity was evaluated again as described above. To evaluate the sustained release of ERY from the PCL/HNT fiber, serial cultivation was examined as follows. The fresh prepared PCL/HNT and ERY fibers were first placed on the inoculated growth media, and the antibacterial effect was evaluated after 24 h of cultivation at 37 ◦C. Then, these same fibers from the grown bacterial cultures were replaced with fresh ones that were again cultivated and considered. This procedure was repeated for 5 days.

#### *2.7. Mechanical Property Measurement*

Mechanical properties were tested in uniaxial tension at a crosshead speed of 5 mm·min−<sup>1</sup> and ambient temperature of 22 ◦C using a Zwick Roell Z010 (Zwick-Roell, Ulm, Germany) equipped with a 10 kN load cell. Six specimens of rectangle shape (approximately 30 × 5 mm2) were tested for each type of material, and the averages and

standard deviations were determined. The thickness of the specimens was measured with a micrometer.

#### *2.8. Thermogravimetric Analysis*

Thermal stability was determined by thermogravimetric analysis (TGA) using a Q1500 D instrument (from MON Budapest, with TA Universal Analysis software). Each sample (100 mg) was heated from 30 to 600 ◦C at a heating rate of 10 ◦C/min in the presence of air with a flow of 50 mL/min. The corresponding weight loss was recorded as a function of temperature.
