Cellulose Acetate Fibers With Infiltrated ZnO Nanocrystals: Activation of Antibacterial Properties Against Acne vulgaris by Oxygen Plasma Treatment

ABSTRACT

In this work, we developed biodegradable cellulose acetate (CA) fibers infiltrated with ZnO nanocrystals for the Acne vulgaris (AV) treatment. The antibacterial effect (through Zn²⁺ release) was activated upon oxygen plasma treatment. CA fibers (fiber diameter ≈ 2 µm) were produced via centrifugal spinning, followed by ZnO infiltration (up to 8 wt% of Zn content) using Vapor Phase Infiltration (VPI) with varying deposition cycles. As CA fibers are naturally hydrophobic, oxygen plasma treatment was applied to turn them hydrophilic and to improve skin wetting. The combination of these steps is presented for the first time. Plasma treatment introduced oxygen‐containing functional groups on the CA surface, significantly improving wettability. Scanning electron microscopy analyses of treated fibers showed no morphological damage. Detailed characterization (using X‐ray photoelectron spectroscopy, Raman spectroscopy, energy dispersive X‐ray fluorescence, X‐ray diffraction, tranmission electron microscopy) focused on fibers with 32 and 128 VPI cycles, revealed that plasma partially oxidized and etched the surface, affecting the ZnO distribution. Plasma‐modified fibers, unlike untreated ones, exhibited antibacterial activity against AV‐causing bacteria (Cutibacterium acnes and Staphylococcus epidermidis), creating significant inhibition zones (up to 5 mm), demonstrating their potential as promising therapeutic modality. Overall, plasma treatment enabled effective surface functionalization, producing antibacterial fibers with enhanced surface properties suitable for application in AV management.

We present biodegradable cellulose acetate fibers infiltrated with ZnO nanocrystals for Acne treatment. Fibers produced by centrifugal spinning were vapor‐phase infiltrated to contain uniform ZnO nanocrystals. Oxygen plasma treatment enhanced the fibers´ wettability. Structural and chemical analyses confirmed that oxygen plasma induces surface functionalization and promotes uniform ZnO redistribution. The resulting fibers exhibited strong antibacterial activity against acne‐causing bacteria.

1. Introduction

Acne vulgaris (AV) is a dermatological disease that affects a wide range of the population, especially young adults. Its occurrence is primarily associated with hormonal or dietary factors that can lead to overgrowth of bacteria on the skin and, in this way, contribute to the development of dermatological diseases [1, 2]. One example is the overgrowth of Cutibacterium acnes, which thus becomes a pathogenic bacterium [3, 4], triggering greater sebum production, eventually leading to AV [5, 6]. Besides Cutibacterium acnes, several other bacterial species have been related to the AV development. Among them, Staphylococcus epidermidis should be listed. Although the exact role of Staphylococcus epidermidis in AV is not fully understood, studies have suggested that it may play a significant role in determining the severity of AV [7, 8].

Depending on the severity, AV can be treated orally or topically. For mild forms, topical agents such as retinoids, salicylic acid, and azelaic acid are commonly used. However, these are often required only in higher concentrations or in combination with other agents [9, 10]. Moreover, another disadvantage of their use is that they cause skin dehydration, itching, burning, and/or dryness [11, 12].

Zinc oxide (ZnO) nanoparticles (NPs) are promising as a therapeutic alternative for the management of AV [13]. ZnO exhibits antibiotic activity, upon release of Zn²⁺ and by forming reactive oxygen species formed during UV‐activated photocatalytic processes. By utilizing NPs, due to the enhanced surface area, the antibiotic activity is significantly enhanced [14]. Despite concerns about the potential penetration of NPs into the bloodstream through the skin, many studies have reported that even during long‐term topical administration, NPs do not penetrate beyond the stratum corneum and have not been associated with any adverse effects on the skin [15, 16, 17].

The comfort of handling and storage makes the recently developed fiber‐based cosmetic masks more attractive than water‐based cosmetic products. These fiber masks are also sold in dry form and therefore do not require chemical preservatives [18, 19, 20, 21]. They offer plenty of advantages, including considerable high porosity and surface area to volume ratio. These fiber‐based soluble masks disintegrate rapidly upon contact with an aqueous medium, allowing for rapid and effective release of active ingredients on the skin [22]. Electrospinning is a commonly used method for the fiber production, but an alternative method, centrifugal spinning, offers a more environmentally friendly approach. It can process comparably more viscous solutions, thus reducing the need for organic or aqueous solvents, thereby reducing also health, environmental, and economic burdens [23]. Moreover, this simple technology, inspired by cotton candy machines, enables easy and efficient handling of the resulting fibers and does not require high voltage, offering a considerably higher production rate than electrospinning [24, 25].

The embedding of ZnO NPs into fibers can be achieved via three fundamentally different approaches: (i) by direct addition of NPs into the spinning solution (thus prepared using a one‐step spinning process), (ii) post‐deposition from a liquid solution, (iii) infiltration of ZnO from the vapor phase.

Regarding the first approach, only two studies have described the preparation of fibers combined with ZnO NPs, which are effective against the bacteria causing AV. In the first paper, we reported the preparation of biopolymeric fibers using centrifugal spinning blended with ZnO NPs under different concentrations ranging from 0.03 to 4.5 wt% [19]. However, Jeong and Oh [26] prepared polyvinyl alcohol (PVA) fibers using electrospinning blended with ZnO NPs from 1 to 7 wt%. However, in both these cases, NPs were embedded directly into the fibers during the manufacturing process, and only part of them protruded onto the surface, this having decreased their availability for the external environment. The nature interaction between ZnO NPs and the fibers is primarily affected by the surface properties of both components, especially their charge, which influences the strength and type of interaction, such as electrostatic attraction or repulsion, and also affects the overall distribution of NPs within the fiber surface [27, 28].

The second approach involves the deposition of ZnO NPs onto the already prepared fibers using a dip‐coating method. This method is predominantly used for hydrophobic fibers and considerable limitations lie in the tendency of NPs to aggregate on the fiber surface. To the best of our knowledge, there are no available studies showing utility of this approach for the preparation of fibers with ZnO for the treatment of AV. However, it should be mentioned that studies providing positive data about fibers antibacterial activity against bacteria Staphylococcus aureus and Escherichia coli [29, 30].

The third approach, resulting in direct infiltration of ZnO NPs into polymer fibers, exploits vapor phase infiltration (VPI). VPI enables the most stable connection of NPs to fibers among all available techniques. VPI is a solvent‐free vapor phase process that allows the diffusion of vaporized metal‐containing precursor molecules into the polymeric matrix and their binding to polymer chains, forming an inorganic–organic hybrid material [31, 32]. This method is similar to atomic layer deposition, which typically deposits nanometers of metal oxide layers onto the surface of a material, but with extended exposure times to allow in‐diffusion during the process [33, 34, 35]. This process was first demonstrated in a study that infiltrated zinc, titanium, and aluminum oxides into spider silk, which significantly increased the toughness of the resulting material [31]. Improvements of electronic and optical properties, as well as photocatalytic performance, were also observed upon VPI [36, 37]. Another benefit of VPI is its ability to enhance antibacterial properties of materials by uniformly infiltrating metal oxide nanostructures, such as TiO₂, Al₂O₃, and ZnO into polymer chains [28].

Our recent study [28] shows that VPI, applied to hydrophilic PVA fibers, grows sub‐surface ZnO nanocrystals that, after 32 or more infiltration cycles exhibit antibacterial activity against AV‐causing bacteria. However, the use of hydrophilic fibers possesses certain disadvantages, in particular an increased susceptibility to microbial degradation, especially in humid environments, which may limit their long‐term durability. Moreover, in biomedical applications, significant changes in the structure of hydrophilic polymers may adversely affect and compromise the biocompatibility of the resulting intermediates. The use of hydrophobic fibers can help to mitigate these disadvantages [38, 39]. On the other hand, the use of the hydrophobic fibers results in a polymer that is insoluble in aqueous environments, hampering the release of active substances (often in the order of several hours), hence, affecting the therapeutic efficiency. To overcome this limitation, plasma, laser, or chemical treatment can be used to introduce easily hydrolysable groups on the polymer surface, which mediate better partial solubility of the resulting polymer fibers [40, 41].

While chemical treatments are simple in processing, in general they possess disadvantages due to the use of hazardous chemicals (e.g., sodium hydroxide, hydrochloric acid, phosphoric acid, etc.) whose toxicity and environmental concerns represent substantial drawbacks of this surface modification method [42, 43]. In contrast, laser‐mediated surface modification of polymers enables quick, precise, and selective treatment of small areas without affecting the surrounding material. However, a limitation of this method is that its effectiveness is governed by the chemical structure and thermal stability of the polymer, with some materials prone to degradation or fragmentation under laser irradiation [44, 45].

Low‐temperature (cold) plasma is widely used for processing of various materials for various applications, including biomedical ones. The main benefit is that the materials can be processed at room or at only slightly elevated temperature, and it modifies only the very surface of a material without causing harmful side effects to its bulk structure/chemistry, as demonstrated by comparing with alkali treatment of 3D‐printed polylactid acid scaffolds [46]. Moreover, its use offers an elegant alternative to conventional sterilization techniques [47, 48, 49]. In particular, oxygen plasma is widely employed to selectively introduce chemically reactive polar functional groups onto material surface enabling the change of hydrophobic surfaces into hydrophilic ones. Besides chemical functionalization, the oxygen plasma treatment also leads to material etching, especially in the case of organic materials (polymers). The extent of surface oxidation and etching depends not only on the process parameters [46, 50], but also on polymer properties, their chemical composition and crystalline structure [51]. Kusumocahyo et al. [52], confirmed that oxygen plasma treatment of cellulose acetate membrane for 60 s decreases the water contact angle from 56° below 20° for the highest discharge power thanks to increased surface oxidation.

Plasma treatment can be performed in low or atmospheric pressure discharges. Although, the atmospheric pressure systems might be preferred due to their initial low‐cost arrangements, low‐pressure plasma offer more uniform chemical functionalization over large areas, easier control of the gas impurities, and interplay of chemical and physical processes thanks to higher energy of ions impinging on the surface. Kikani et al. [53], demonstrated that treatment of polyethylene with the low‐pressure dry‐air plasma resulted in the surfaces with higher surface energy and slower aging compared to the atmospheric pressure air plasma.

Currently, only a limited number of studies [54, 55] have report the preparation of cellulose acetate (CA) fibers containing ZnO nanoparticles for antibacterial application. In these studies, the fibers were prepared by electrospinning, where ZnO nanoparticles were incorporated either directly into the spinning solution (prior to the fiber formation) or deposited onto the fiber surface after their fabrication. However, these approaches have several limitations. In particular, they often lead to a non‐homogeneous distribution of ZnO within the fibrous structure. Moreover, the nanoparticles are typically incorporated through physical embedding within the polymer matrix, which may result in their undesirable release over time [54, 55]. In contrast, using Vapor Phase Infiltration, ZnO is introduced directly into the pre‐formed CA fibers. This process enables partial chemical integration of ZnO with the polymer matrix, more homogeneous distribution of nanocrystals and improved stability [56, 57]. Subsequent plasma treatment can then be applied to selectively activate the resulted fibers, tailoring their functionality for specific applications. Therefore, this study focuses on an alternative strategy that enables a more homogeneous infiltration of ZnO nanocrystals within CA fibers by combining VPI with plasma treatment.

To the best of the authors´ knowledge, no work has been published to date that combines VPI of polymeric fibers with subsequent plasma treatment. The combination of these two techniques is significant: VPI allows for the incorporation of ZnO into the fibers, while plasma treatment alters the fiber structure, facilitating its transformation. Therefore, this work represents a novel approach, not only for treatment of AV but also for other potential applications.

This work aims to modify hydrophobic CA fibers, which are commonly used in skin treatment and can thus be considered biosafe and as carriers for antibacterial application [58, 59, 60, 61, 62, 63]. They were employed as matrix for the therapeutic AV‐treatment. At first, CA fibers were prepared by centrifugal spinning. Following this, the CA fibers were infiltrated with ZnO by VPI (1–128) cycles and then modified by low‐pressure oxygen plasma treatment. The modified fibers, with and without plasma treatment, were analyzed using several characterization techniques, also in comparison to a reference plasma‐treated planar CA films on glass substrate. The results revealed that plasma treatment increased the hydrophilicity of fibers and led to the efficient activation of their antibacterial properties. Taken together, this work represents a pioneering contribution, demonstrating the effect of the plasma treatment on ZnO‐infiltrated CA fibers and shows their greatly promising potential for AV treatment.

2. Results and Discussion

2.1. Morphological and Elemental Characterization

Figure 1 shows scanning electron microscopy (SEM) images of the CA fibers after infiltration of ZnO with various number of VPI cycles, before and after plasma treatment. As a reference, CA fibers without infiltration are shown in the first row. According to the images, neither the plasma treatment nor the ZnO infiltration caused visible damage to the fibers or melting. As shown in Table S1, the fiber diameter remained at approximately 2 µm, determined as the median value based on 30 measurements for each cycle, even after the VPI process. Likewise, the plasma treatment did not apparently change the fiber diameter. No clear dependence of the mean fiber diameter on the number of VPI cycles or plasma treatment was observed among the fibers by SEM analysis. The large standard deviations indicate substantial variability in fiber diameters, suggesting that any dimensional changes induced by ZnO infiltration or plasma treatment are negligible with respect to those large fiber diameters.

FIGURE 1.

SEM images of as‐prepared CA fibers and ZnO‐infiltrated CA fibers, produced by varying numbers of VPI cycles (between 1 and 128), before and after oxygen plasma treatment. All scale bars represent 20 µm.

Cross‐sectional SEM analyses of the fiber/ZnO interface were performed to obtain a detailed overview of the ZnO morphology. Figure 2 shows SEM images that were acquired from CA fibers with 128 VPI ZnO at low and high magnifications, before and after plasma treatment, using secondary electron (SE) and backscattered electron (BSE) detectors. In addition, very high magnification SEM images of the ZnO/fiber interface after 128 ZnO VPI cycles before and after oxygen plasma treatment are shown in Figure S1. As one can see, the CA fibers are uniformly infiltrated with ZnO, and the layer remains strongly attached even to fractured fiber surfaces. This observation indicates that the ZnO has indeed infiltrated the fiber structure rather than solely forming a surface layer (coating) as atomic layer deposition (ALD) would do. Indeed, coatings deposited by conventional ALD would tend to delaminate from broken fibers. Following the plasma treatment, the ZnO layer continues to exhibit a uniform infiltrated zone, indicating that the plasma treatment preserves the infiltration status of ZnO.

FIGURE 2.

Cross‐sectional SEM images of CA fibers after 128 ZnO VPI cycles, before and after oxygen plasma treatment, at both low and high magnifications, using SE and BSE detectors. ZnO is in all cases infiltrated within the polymer fiber body.

The infiltration of ZnO into the CA fibers was further evaluated using energy dispersive X‐ray fluorescence (EDXRF) by determining the amount of Zn as a function of the number of VPI cycles. As shown in Figure 3, the ZnO content increased with the number of VPI cycles. This increase exhibited a non‐linear trend, typical for the VPI process as presented in previously published studies [28, 64, 65], indicating that Zn is being infiltrated into the fibers. In contrast, if ZnO was coated as a layer onto the fibers, linear growth of ZnO would be expected, which would be typical of the ALD.

FIGURE 3.

The Zn concentration (wt%) in ZnO‐infiltrated CA fibers with varying VPI cycles, measured by EDXRF.

The sequence of plasma treatment after VPI was strategically selected. Performing the plasma surface functionalization prior to VPI would modify the fiber surface chemistry in an uncontrolled and heterogeneous way. Moreover, the plasma treatment prior to VPI would alter their rheological properties and disable accurate TGA measurements required for reliable VPI process. Furthermore, prior plasma treatment could hinder the subsequent penetration of the ZnO precursor due to altered density of the polymer, leading to uneven ZnO distribution. Based on these considerations, the sequence used was chosen accordingly.

Two representative samples (32 and 128 ZnO VPI cycles) were selected for detailed characterization and discussion to investigate the effects of plasma treatment in greater detail. The selection was based on quantitative and qualitative observations: 32 VPI corresponds to the onset of observable antibacterial activity, while 128 VPI represents a sample with the highest Zn²⁺ content. This approach ensures that the samples are representative and suitable for systematically exploring the relationship between surface modification, structural and vibrational characteristics and antibacterial activity. They effectively represent the range of functional properties and allow a systematic investigation of the relationships between surface modification, structural and vibrational characteristics, and antibacterial activity.

High‐resolution TEM, as shown in Figure 4, was used to analyze the CA fibers after 32 and 128 ZnO VPI cycles, both before and after plasma treatment. In Figure 4, it can be observed that CA fibers with 32 ZnO VPI cycles exhibit some scattered ZnO nanocrystals on the surface of the fibers. The reason is that, although the VPI process targets infiltration of polymers, the same chemical functionalities of the polymer are present also on the surface of the fibers, which causes an ALD coating in parallel to the infiltration, which is difficult to avoid [66]. Upon detailed SEM analyses (Figures 1 and 2 and Figure S1), we did not see any significant changes in the thickness of the ZnO infilitrated layers before and after the plasma treatement. However, upon detailed inspection of samples by TEM a more disordered and less uniform ZnO nanocrystal arrangement can be seen, suggesting that part of layer was removed due to the plasma exposure. However, this overall did not change the thickness significantly.

FIGURE 4.

TEM images of CA fibers with ZnO after 32 and 128 VPI cycles, before and after oxygen plasma treatment. The green line indicates the layer of ZnO nanocrystals.

Figure 5 shows the presence of ZnO on the CA fibers, as confirmed by TEM in scanning transimisson electron microscopy (STEM) mode and in STEM with energy dispersive X‐ray spectroscopy mode (STEM‐EDS) analyses conducted before (BP) and after plasma (AP) treatment. In all cases, carbon dominates the fiber core, whereas zinc and oxygen are concentrated at the surface, confirming successful ZnO coating. As shown in Figure 5, a homogeneous ZnO nanocrystalline layer was formed onto the CA fibers with 32 VPI cycles before plasma treatment. For fibers with 128 VPI cycles, the ZnO nanocrystalline layer appears more pronounced compared to 32 VPI cycles, forming a dense and continuous coating observable before plasma treatment. After plasma exposure, this layer becomes highly porous and fragmented, indicating a pronounced restructuring of the ZnO morphology. This indicates that higher ZnO loadings still undergo surface modification under plasma, but retain a substantial ZnO presence on the fibers, which is also evident in Figure 4.

FIGURE 5.

STEM and STEM‐EDS elemental maps of the CA fibers with ZnO after 32 and 128 VPI cycles, before (BP) and after oxygen plasma treatment (AP). Elemental distribution maps of carbon (C), oxygen (O), and zinc (Zn) are presented along with their overlapped map.

2.2. Structural and Chemical Characterization

Figure 6a shows X‐ray diffraction (XRD) patterns of CA fibers after 32 ZnO VPI cycles, before and after plasma treatment. The presence of crystalline ZnO with a hexagonal wurtzite structure is indicated by the diffraction peak corresponding to the (101) plane, observed in the XRD pattern within the 2θ range of 35°–39°. However, the peak is not sharp, suggesting a lower amount of ZnO within the fibers. After plasma treatment, a slight shift accompanied by peak narrowing indicates modifications in the ZnO lattice, likely resulting from changes in lattice strain or an increased defect concentration [67].

FIGURE 6.

XRD characterization of CA fibers with ZnO before and after oxygen plasma treatment. Main ZnO diffraction peak in the range 35°–39°, corresponding to the (101) plane, is shown for (a) 32 VPI ZnO and (b) 128 VPI ZnO, and the second main diffraction peak in the range 30°–35° corresponding to the (100) plane, is shown for (c) 128 VPI ZnO.

The main peaks from the XRD patterns of CA fibers infiltrated with 128 VPI ZnO cycles are shown in Figure 6b,c, The XRD peaks in the 2θ ranges of Figure 5b 35°–39° (corresponding to the (101) plane) and Figure 5c 30°–35° (corresponding to the (100) plane) show clearly defined diffraction peaks. Compared to the 32 VPI ZnO sample, higher resolution and a stronger signal were observed, confirming the presence of crystalline ZnO with a hexagonal wurtzite structure. Plasma treatment caused subtle shifts in peak positions and changes in intensity, indicating structural disruptions in the ZnO lattice, as observed also for fibers with 32 VPI ZnO [67]. Damage to the originally uniform ZnO layer by plasma discharge is also clearly visible in TEM images (Figure 3).

Figure 7 shows Raman spectra of CA fibers infiltrated with 32 VPI ZnO and 128 VPI ZnO before and after plasma treatment. The retention of the characteristic Raman bands of ZnO at ∼384 and ∼434 cm⁻¹ indicates that the oxide was not degraded or removed under the applied plasma treatment conditions, confirming its structural stability [68, 69].

FIGURE 7.

Raman spectra of CA fibers infiltrated with ZnO before and after oxygen plasma treatment for (a) for 32 VPI and (b) 128 VPI cycles, where ZnO dashed lines correspond to main ZnO peaks at 384 and 434 cm⁻¹ and red dashed lines indicate positions of new CA chemical groups generated at the interface between ZnO and CA within ZnO‐infiltrated CA fibers.

Regarding CA itself, the characteristic vibrational bands in the 500–1800 cm⁻¹ range were observed in CA fibers after ZnO VPI, both before and after plasma treatment. These spectra are consistent with those reported in previous studies [70, 71, 72, 73, 74].

However, several differences were observed between the spectra of before and after plasma treatment of the CA fibers with 32 and 128 VPI ZnO. For CA fibers with 32 VPI ZnO (Figure 7a), the plasma treatment led to a decrease in the peak intensity at ∼602 cm⁻¹, which can be attributed to C═O bending vibrations and indicates chemical changes related to acetyl groups [70, 71]. In addition, a band at ∼702 cm⁻¹, associated with skeletal vibrations of the pyranose ring, appeared after plasma treatment. A band at ∼740 cm⁻¹, not typically assigned to CA, disappeared after plasma treatment. This peak may have originated from impurities or secondary interactions rather than intrinsic CA vibrations. The band around ∼881 cm⁻¹, characteristic of CA, also vanished after plasma treatment, indicating reduced C─O─C stretching vibrations [70]. Furthermore, the peak at ∼1045 cm⁻¹, corresponding to the stretching mode of the C─O single bond, disappeared [75]. Distinct new peaks emerged at ∼1656 and ∼1700 cm⁻¹, which can be assigned to C═O stretching vibrations of acetyl groups [70, 71].

In contrast, CA fibers with 128 VPI ZnO (Figure 7b) exhibited only minor spectral changes after plasma treatment. The overall profile remained largely unchanged, except for the appearance of two bands at ∼1660 and ∼1700 cm⁻¹, corresponding to C═O stretching of acetyl groups, indicating a less pronounced chemical modification [70, 71].

This indicates that higher ZnO loading results in more limited structural alterations of the CA matrix, while still promoting the formation of carbonyl functionalities at the ZnO/CA interface. This behavior highlights the synergistic effect of plasma discharge on the ZnO layer, which acts as a catalyst, selectively oxidizing the CA surface in contact with ZnO and promoting the generation of additional C═O groups within the polymeric matrix [76, 77, 78].

To gain a deeper understanding of the chemical changes induced by the plasma treatment, the surface composition of the fibers was examined using EDXRF and XPS techniques.

Both analytical methods confirmed a remarkable reduction in the surface Zn content after the plasma treatment. However, it should be noted that the two techniques probe different information depths. EDXRF provides elemental analysis over a depth of several micrometers and the results therefore include the composition of both the surface itself and the bulk material of the fibers [79]. According to EDXRF analysis (Table S2), the Zn level decreased after plasma treatment by ∼72% in the fibers with 32 VPI ZnO (from 2.49 to 0.69 wt%) and by ∼19% in the fibers with 128 VPI ZnO (from 7.92 to 6.44 wt%). The decrease in the Zn content can be explained by the destructive effect of plasma on the surface layers of CA fibers. The original homogeneous ZnO layer is destroyed by the incident reactive particles (ions, electrons), it is gradually sputtered off of the surface, thereby reducing the total zinc content in EDXRF spectra. This is in good correlation with the results observed from TEM, STEM‐EDS, and XRD analyses (Figures 4, 5, and 6).

Consistently, the highly surface‐sensitive XPS analysis (with analytical depth of approx. 10 nm), indicated a similar decrease in the Zn content, confirming that the reduction also occurs at the very surface of the fibers. Table 1 shows the atomic percentages (at%) of the main elements. In addition to the decrease in Zn, there is also a decrease in oxygen. Both indicate the destructive effect of plasma on the outer side of ZnO nanocrystalline layer. This is more obvious seen in Figure 8, which shows XPS high‐resolution spectra of Zn 2p and O 1s. The position of the Zn 2p^3/2 corresponds to ZnO (BE = 1022 eV, ΔBE_(3/2‐1/2) = 23 eV). [80] These values and the shape of the Zn peaks themselves do not change after the application of oxygen plasma, which confirms that the chemical nature of the ZnO nanocrystalline layer itself remains unchanged. In contrast, the oxygen spectrum O1s undergoes a significant change. Two main contributions are evident in the oxygen spectrum, where a less intense, narrow peak in the region of 529–531 eV can be assigned to oxygen bound as a metal oxide (ZnO) and then the contribution of a broad peak around 532.5 eV, which belongs to organically bound oxygen in the CA molecule (mainly acetate groups) [80, 81]. After the plasma treatment of both reference samples, a significant decrease in oxygen bound to zinc is observed (O2/O1 ratio Table 1). This indicates a thinning of the ZnO nanocrystalline layer on CA fibers and confirms the results obtained from TEM analyses shown in Figure 4, reflecting changes in their morphological structure.

TABLE 1.

Atomic percentage (at%) of C, O, and Zn from XPS spectra of the CA fibers after 32 and 128 ZnO VPI cycles and after oxygen plasma treatment (*O2/O1 ratio from contributions of O 1s spectra).

at%	C	O	Zn	O2/O1*
CA fibers + 32 VPI ZnO/ before plasma	62.0	33.1	4.8	1:8
CA fibers + 32 VPI ZnO/ after plasma	71.0	26.6	2.3	1:23
CA fibers + 128 VPI ZnO/ before plasma	57.6	32.0	10.4	1:3
CA fibers + 128 VPI ZnO/ after plasma	67.8	25.8	6.4	1:7

FIGURE 8.

High‐resolution XPS spectra of Zn 2p and O 1s recorded from CA fibers with 32 and 128 ZnO VPI cycles before and after oxygen plasma treatment.

Unfortunately, the C 1s carbon spectrum from these CA polymer matrices (in particular for fibers with 128 VPI ZnO) has a low intensity due to the overlay of the ZnO layer. Therefore, it is not suitable for high‐resolution analysis, and in particular, not for the assessment of the individual contribution of CA bonds.

Hence, the spin‐coated planar CA films on glass substrates, as described further, were employed, which provide a much better signal to noise ratio for both states—before and after plasma treatment. Moreover, planar CA films enable reliable measurement of Water Contact Angle (WCA), as described further. WCA cannot be reliably measured on bulkier fibrous networks, where strong capillary interaction would influence the wetting behavior. Thus, this justifies the need to provide reference measurements also on planar films.

It is characteristic of polymer samples treated with plasma that after a certain period of time they tend to regenerate their properties to their original state. This negative trend is known as the aging effect [82]. As part of the assessment of the aging effect, it was decided to perform analytical tests on the samples and evaluate any changes in the material composition. The samples were placed in polyethylene bags and stored dry and dark at ambient temperature. After a year of aging, the elemental composition and Hi‐Res analysis of zinc and oxygen atoms were determined by XPS. The results are presented in Table S3 and Figure S2. From the elemental analysis it is evident that after a year of storage the chemical composition in both cases 32 and 128 VPI changed very minimally (in the order of percentage units). A slight decrease in oxygen content and a related increase in carbon and zinc can be observed. Within the framework of Hi‐res elemental analysis, it was found that the Zn spectra are practically unchanged, thus aging is only manifested in the polymer part of the nanofibers. From the oxygen spectra, it is evident that there is a decrease in the contribution of organically bound oxygen and thus a change in the O1/O2 ratio. This phenomenon is expected and is probably related to post‐plasma reactions of unstable oxygen groups on the surface of the polymer fibers and the subsequent formation and release of low‐molecular substances [83]. The overall evaluation concludes that the storage of samples and their aging do not have a significant effect on the material composition of the samples.

2.3. Plasma Treatment of Planar Reference CA Films for Surface Analysis

To investigate the effect of plasma treatment on the polymeric CA material by surface analytical techniques that are not entirely applicable and/or sufficiently accurate for bulky fibrous structures, planar reference samples of CA films were intentionally prepared. For this purpose, pure CA solution (16 wt%) was deposited on glass substrates using a spin‐coating technique and dried. This approach allowed to boost the number of characterization techniques, not only XPS analysis and Raman spectroscopy, but also, very importantly, WCA measurements, which require a flat and smooth surface to accurately assess surface wettability and infer the underlying chemical modifications.

In terms of XPS results, the Hi‐res C 1s reference spectrum of CA was analyzed using 5 contributions (C1–C5) according to the model from the organic polymer database [81]. The contributions C1 and C5 are related to the presence of acetate groups, while the contributions C2, C3, and C4 represent different carbon groups in the cellulose molecule. A detailed description including the assignment of the C1–C5 peaks is given in Figure S3. A high level of agreement was found in the shape, position, and intensity of the individual contributions (Figure 9). After plasma treatment of the samples, significant differences in the shape of the C 1s spectrum and the intensity of the individual contributions can be seen. Specifically, the content of acetyl groups (C5) decreases, supporting the notion of plasma‐induced degradation and removal of surface functional groups. Additionally, the C3 peak, associated with the carbon ring of glucopyranose, also decreases, indicating damage to the polymer chain backbone. The observed increase in the C1 component may be related to the formation of new oxygen‐depleted carbon species, while the increase in the C4 group corresponds to possible C═O carbonyl functionalities. However, the appearance of new carboxyl groups (C─O─O) can also be assumed, and their presence would be indicated by a slight broadening to higher energies of the spectrum in the C5 region, as seen also by Kusumocahyo et al. [52] A detailed assignment and quantitative content of the individual XPS C1s peaks for the CA sample (spin‐coated CA film) before and after plasma treatment is shown in Table S4. Overall, these results corroborated general fact that formation of polar oxygen groups (such as C─O─O, C═O) leads to an increase of their hydrophilicity and a decrease of water contact angle, as also demonstrated by Kusumocahyo et al. [52]

FIGURE 9.

High‐resolution C 1s XPS spectrum of planar CA film before (left) before and after oxygen plasma treatment (right).

Furthermore, Raman analysis was performed to assess the plasma effect on planar CA films spin‐coated onto glass. As shown in Figure S4, the Raman spectra remained unchanged during plasma treatment, indicating that the plasma does not affect the deeper layers of the polymer. This is consistent with the fact that the Raman analytical depth is much greater than the plasma penetration depth. The exact affected depth of treatment depends on various parameters such as plasma parameters (treatment time, power, and oxygen flow rate) and the polymer crystallinity/density [84], and usually is in the range of the tens of nm, in the case of a porous structure, this depth may be greater. However, Raman signals were detected using a 633 nm red laser at depths of analysis up to 1–2 µm, depending on the polymers used [85]. At the same time, it should be noted that the ability to detect changes in Raman spectra is also related to the structure of the sample. In the case of planar samples, where the effects of plasma are manifested only on the surface at a small depth, the chance of detecting these changes is very small. Most of the signal in the spectra comes from the untreated internal mass. However, if the sample is porous with a much larger structured surface, the probability of detecting surface changes increases significantly. This is also the reason why oxidation changes can be observed on plasma‐treated fibers (Figure 7), while not for compact planar samples. This effect is generally valid also for other “deep‐range” analytical techniques, such as EDX, XRD, or ED XRF, and whose results can then be applied to “short‐range” technique (XPS) with certain limitations.

To further assess surface modifications, changes in wettability were examined by measuring the WCA on CA thin films before and after plasma treatment. The contact angle of the untreated CA film was 57.1° (±2.1°) and decreased to 15.2° (±2.6°) after plasma treatment. In general, it can be stated that the value of the water contact angle is related to the surface properties of the material, its chemical composition and structure. A decreasing value of the contact angle indicates a stronger hydrophilicity of the material. The increased hydrophilicity in the present case is mainly caused by the chemical change of the polymer chain and the formation of new polar groups increasing the hydrophilic character of the material, which was demonstrated by XPS (Figure 8). Increased wettability can therefore also be expected for CA fibers with 32 and 128 ZnO VPI that were exposed to oxygen plasma. In these fibers, after plasma treatment, both structural changes, increased roughness or surface distortion, visible in TEM images (Figure 4), and oxidative chemical changes, detected by Raman and XPS (Figures 7 and 8), were observed. A similar trend was also observed by Kusumocahyo et al. [52]

2.4. Antibacterial Activity

To provide insights into the effect of the oxygen plasma treatment on biological activity of ZnO‐infiltrated CA fibers, their antibacterial activity was tested against acne‐associated bacteria.

As shown in Table S5, Figures S5 and S6, no inhibition zones were observed for C. acnes and S. epidermidis administered with CA fibers not treated with plasma. The absence of antibacterial activity is likely due to the hydrophobic nature of the CA fibers and the firm embedding of ZnO nanocrystals within the polymer matrix, which may involve partial hybridization between ZnO and CA. As a result, ZnO nanocrystals were not released into the medium, which is primarily water‐based, and thus no inhibition of S. epidermidis at 24 h or C. acnes at 48 h was observed.

Nevertheless, plasma treatment of the sample with infiltrated ZnO induced significant changes that led to detectable antibacterial activity against C. acnes and S. epidermidis. This activity became evident for fibers with 32 cycles of ZnO VPI, where inhibition zones began to appear for both bacterial strains, as shown in Table S5, Figures S5, and S6. This response can be plausibly attributed to plasma‐induced morphological and surface changes, primarily affecting a chemical structure of CA fibers means (i.e., disruption of the polymer surface and formation of new oxygen‐containing compounds, leading to changes in the fiber's hydrophilicity, as described above) These modifications facilitated the release of ZnO nanocrystals from the ZnO‐infiltrated CA fibers upon contact with aqueous agar, thereby imparting the antibacterial activity.

The inhibition zones of samples after plasma treatment are shown in Figure 10, and the corresponding mean values ± SD are provided in Table S5. Figure 10 shows that CA fibers infiltrated with 32 ZnO VPI cycles exhibited an inhibition zone of approximately 2 mm, whereas fibers with 64 ZnO VPI showed zones of around 5 mm for both C. acnes and S. epidermidis. Fibers with 128 ZnO VPI demonstrated inhibition zones of 5 and 5.40 mm for C. acnes and S. epidermidis, respectively. These findings suggest that the antibacterial activity against C. acnes reaches a plateau after 64 VPI, while inhibition of S. epidermidis continues to increase with higher cycle numbers.

FIGURE 10.

The inhibition zone (mm) for ZnO‐infiltrated CA fibers with different VPI cycles, after oxygen plasma treatment. Bars represent mean values of inhibition zone diameters (mm). Error bars indicate mean ± standard deviation (SD) calculated from three independent measurements (n = 3). “X” denotes no detectable inhibition.

This behavior is consistent with the ZnO dissolution mechanism reported in our previous studies [19, 28]. ZnO undergoes partial dissolution when in contact with the agar medium, leading to the release of Zn²⁺ ions. These ions are able to diffuse into the surrounding agar, where they interact with bacterial cells and are responsible for the observed antibacterial activity. The extent of bacterial growth inhibition therefore correlates with the ZnO content in the fibers, as higher ZnO loading results in increased Zn²⁺ release and larger inhibition zones (Figures S5 and S6).

As previously mentioned, CA offers several significant advantages, including a hydrophobic nature, which reduces microbial colonization compared to hydrophilic polymers. In addition, CA has been demonstrated to possess good thermal and hydrolytic stability and chemical resistance [58]. Therefore, this polymer has great potential as a fiber matrix and in combination with ZnO for antibiotic applications.

In our previous study [28], in which ZnO was infiltrated into hydrophilic fibers, the antibacterial activity started from 16 VPI cycles for S. epidermidis and 32 ZnO VPI cycles for C. acnes. In this study, even though the same number of VPI cycles was used, antibacterial activity observed against both bacteria started at 32 ZnO VPI cycles only when the plasma treatment was included. Overall, the antibacterial activity was detected solely in plasma‐treated fibers, highlighting the crucial role of plasma‐induced surface modifications and ZnO. The plasma treatment modifies the fiber surface both chemically and morphologically, increasing its reactivity and enabling the release of ZnO nanocrystals. It is obvious that the combination of plasma‐induced surface modification and the ZnO presence is responsible for the observed antibacterial activity against C. acnes and S. epidermidis, making them an interesting choice for various dermatological applications.

It should be noted that the samples were tested immediately after the plasma treatment and were kept in dark boxes, so photocatalytic effects induced by light can be excluded. The antimicrobial activity of ZnO‐infiltrated fibers may result from several interacting mechanisms. The antibacterial activity, is therefore attributed to the reaction of plasma with ZnO‐infiltrated CA fibers, since plasma‐treated fibers without ZnO showed no antibacterial activity (Figures S5, S6, and Table S5). Plasma treatment can enhance the generation of oxygen‐containing groups on ZnO, which contribute to oxidative damage of microbial cells, including destruction of DNA and protein in the membrane [86, 87].

In addition, the change in surface wettability from hydrophobic to hydrophilic after plasma exposure, may promote the dissolution and release of Zn²⁺ from ZnO nanocrystals. These ions can disrupt cell membrane integrity, allowing deeper penetration into the cytoplasm and subsequently inducing oxidative stress [88].

Plasma treatment can also influence the antibacterial activity by promoting partial release of ZnO nanocrystals from the fiber surface. These nanocrystals can cause direct physical damage to microbial cell walls through close contact, ultimately leading to the membrane disruption or perforation. In addition, bacterial surfaces are negatively charged due to the presence of carboxyl groups, while ZnO nanocrystals typically carry a positive surface charge. This electrostatic interaction destabilizes the bacterial cell wall and facilitates the penetration of ZnO nanocrystals into the cell. This effect is particularly pronounced in Gram‐positive bacteria (C. acnes and S. epidermidis), which lack an outer lipid membrane and are therefore less protected [88, 89]. Furthermore, plasma treatment significantly modifies the surface properties of the material, particularly its roughness (as shown in Figure 4), which can further enhance antibacterial activity [90]. Aging of the ZnO‐infiltrated CA fibers also plays a role in long‐term antibacterial activity, as CA fibers with 128 ZnO VPI after plasma treatment retained antibacterial activity even after 1 year, with approximately a 50% reduction compared to their initial level (Table S6). This indicates that antibacterial activity is retained even after 1 year of storage.

This clearly demonstrates that cellulose acetate fibers with infiltrated ZnO, subsequently plasma‐treated, represent a novel and promising biomaterial for AV treatment. This work is pioneering in nature, and further studies, particularly clinical investigations, will be necessary to fully evaluate its efficacy and safety.

• Understanding the release mechanism is crucial for optimizing controlled Zn²⁺ exposure. It is also important for fully understanding the antibacterial activity, complementing the effects observed in the current study.

• The semi‐quantitative method, lacking detailed growth curves or MIC data, will be complemented by additional in vitro testing in future.

• ZnO nanocrystals may enhance the proliferation and migration of fibroblasts and keratinocytes, both of which are crucial for wound healing [91, 92]. While the current study focuses on the preparation and potential suitability of the material for AV treatment, further evaluation through additional in vitro and clinical testing is planned for future work.

• Scavenger assays will be incorporated in future studies to experimentally verify the contribution of ROS species to the antimicrobial effect of the ZnO‐based fibers, complementing the mechanistic insights discussed here.

• Clinical evaluation on patients with AV will be necessary. While direct clinical evidence for AV is limited, Zn‐containing formulations are widely used in dermatology. Based on their reported effects in other skin conditions, positive outcomes in AV treatment are expected.

3. Summary

The aim of the work was to create CA fibers with infiltrated ZnO nanocrystals, and thus combine the material advantages of CA with the antibacterial effects of ZnO. However, the initially created material did not show any antibacterial activity. The problem lies in the design of the antibacterial assay itself, which is based on the gradual release of the antibacterial agent into the liquid phase and the creation of a measurable inhibition zone. In the case that the sample under investigation interacts poorly with the liquid medium or the antibacterial agent is firmly bound to the substrate, the particles are not released, and the antibacterial effect cannot be determined. The solution to this situation is the disruption of the compact outer ZnO layer on the surface of the fibers and the appropriate chemical modification of the polymer containing incorporated ZnO particles. A device containing a low‐pressure electric discharge generating oxygen plasma was chosen as a suitable procedure for this treatment. The idea of the treatment consisted in bombarding the fibers with high‐energy plasma particles, capable of disrupting chemical bonds in the material, and at the same time oxygen ions and radicals can then functionalize polymer chains. The treated materials were subjected to thorough material analysis after the treatment.

Below the individual partial findings are summarized point by point:

• For ZnO‐infiltrated CA fibers, ZnO nanocrystals, that were previously partially or fully embedded in the polymeric matrix, got partially exposed to the environment as a result from plasma‐induced etching. This was applied to fibers with 32 VPI ZnO and 128 VPI ZnO. Moreover, the ZnO nanocrystalline layer remained on the fibers after plasma treatment, especially on the CA fibers with 128 ZnO VPI cycles.

• Raman analysis showed that plasma treatment had a greater effect on CA fibers with 32 ZnO VPI cycles than on those with 128 cycles. In the CA fibers with 32 VPI ZnO, plasma treatment induced the formation and disappearance of various carbon‐oxygen groups, such as C═O and C─O─C, whereas in the fibers with 128 ZnO VPI cycles, only C═O‐containing compounds were observed. This difference can be explained by the higher ZnO content in the CA fibers with 128 VPI ZnO cycles, which provides better protection to the polymer matrix. While oxygen plasma primarily reacted with the near‐surface region of the fibers, the presence of a ZnO nanocrystalline layer can enhance surface oxidation through a catalytic effect at the ZnO‐CA interface.

• Another information that stems from the Raman spectra of ZnO‐infiltrated CA fibers is that the plasma treatment induced slight changes in the overall spectral shape, suggesting intensified surface oxidation. Similar to TiO₂, nanostructured ZnO exhibits catalytic effects on organic materials under UV irradiation, which is a common component of oxygen plasma [76, 77, 78]. Therefore, the ZnO nanocrystalline layer likely acts synergistically with the plasma, leading to more pronounced oxidation of the CA fiber surface, detectable by Raman spectroscopy.

• In contrast, Raman analysis of planar CA films (on glass substrates without ZnO) showed that plasma treatment induced no detectable changes, indicating that pure CA is unaffected. This suggests that the plasma‐induced changes in the fibers are due to the interaction between ZnO and cellulose acetate.

• Changes caused by the plasma treatment were also observed by XPS, which primarily detects surface modifications within approximately 1–10 nm depth from the surface [51, 93]. During plasma exposure, a reduction in ZnO was observed in fibers with infiltrated ZnO. In contrast, for the planar films, which were prepared to provide a uniform reference surface for better analysis of surface chemistry after plasma treatment, new carbon‐oxygen containing compounds (C═O and C─O─O) formed, original polymer functional groups disappeared, and low molecular hydrocarbon residues were generated.

• The oxidative effects of plasma have a positive effect on increased wettability of the CA polymer. It can be assumed that this will result in both increased hydrophilicity and desired solubility of the polymer matrix, once placed on the skin.

• The antibacterial tests showed that plasma treatment had a significant contribution. These effects were attributed to changes in wettability and plasma interactions with ZnO, which positively influenced the antibacterial activity observed in CA fibers starting from 32 ZnO VPI.

• No pronounced aging effect after plasma treatment was observed even after 1 year of storage, as confirmed for samples with 32 and 128 ZnO VPI cycles. Moreover, the sample with 128 ZnO VPI cycles still exhibited antibacterial activity after 1 year.

4. Conclusion

In conclusion, we demonstrated the fabrication of centrifugally spun CA fibers with infiltrated ZnO applying 1, 2, 4, 8, 16, 32, 64, and 128 VPI cycles. Successful infiltration of ZnO was confirmed by high‐resolution SEM combined with EDXRF, and ZnO nanocrystals were also observed on the fibers by TEM, confirming the presence of ZnO both within the fibers and on their surface.

Subsequently, these ZnO‐infiltrated CA fibers were treated with the oxygen plasma to modify their surface properties. The plasma treatment induced morphological and chemical modification of the polymer component on the fibers surface with a change in the surface‐bound ZnO structure. It can thus be concluded that the oxygen plasma treatment alters the interaction between the polymer matrix and ZnO nanocrystals, which influences the functionality of the resulting composite, including the morphological, chemical, and crystalline structure.

Plasma treatment led to a significant reduction in the ZnO content on fiber surfaces, as confirmed by TEM, STEM, STEM‐EDS, EDXRF, and XPS analyses. This reduction reflects the partial removal of ZnO nanocrystals on the fiber surfaces, highlighting the impact of plasma exposure on the fiber morphology and nanocrystal distribution. XRD analyses confirmed the presence and integrity of the ZnO crystalline structure infiltrated in the fibers. While Raman analysis showed pronounced effects only in ZnO‐infiltrated samples, XPS revealed surface changes in all cases, including samples without ZnO. This led to partial loosening and redistribution of ZnO within the fibers, as well as localized changes in surface from hydrophobic to hydrophilic, which facilitate the release of ZnO and contribute to the observed antibacterial activity.

Furthermore, it was shown that samples with lower numbers of ZnO VPI cycles are less resistant to the plasma treatment than those with higher numbers of cycles, resulting in pronounced material changes, characterized by comparative ratios of various oxygen‐containing compounds.

Antibacterial activity of the CA fibers starting from 32 ZnO VPI cycles after plasma treatment was observed against Cutibacterium acnes and Staphylococcus epidermidis, whereas fibers prior to plasma treatment showed no antibacterial activity. This confirms that plasma treatment enhances the activity of ZnO nanocrystals.

This work is the first to demonstrate differences in antibacterial activity related to oxygen plasma treatment of ZnO‐infiltrated CA fibers, highlighting its influence on the morphological and structural properties and its role in antibacterial activity.

Aging studies conducted after 1 year showed no significant degradation in the surface chemical composition as determined by XPS analysis. In addition, antibacterial testing revealed that the sample with 128 ZnO VPI retained evident antibacterial activity even after 1 year.

This finding shows that the material combination of CA and VPI‐infiltrated ZnO and oxygen plasma represents a promising and effective material with in vitro antibacterial potential for the treatment of AV.

5. Experimental Section

5.1. Centrifugal Spinning

CA with a molecular weight of 123 000 g mol⁻¹, acetone and N,N‐dimethylacetamide (DMAc) were purchased from Sigma‐Aldrich (Czech Republic). An organic solution consisting of acetone and DMAc in a volume ratio of 3:1 was prepared using 16 wt% CA. This solution was gently stirred overnight using a magnetic stirrer at 50°C.

Resulting solutions were subsequently spun by the centrifugal spinning tool Cyclon Pilot G2 (Pardam Ltd., Czech Republic). CA fibers in the form of a bulky polymeric structure, were prepared with the following processing conditions: rotational speed of the spinneret 10 000 rpm, temperature 40°C (±5), and relative humidity 15% (±5).

5.2. VPI

Infiltration of the CA fibers with ZnO was performed in a custom‐made fluidized bed ALD/VPI reactor. In each process, 0.9 g of CA fibers were loaded into a vertical stainless steel reactor column of 250 mm height and 25 mm inner diameter. Diethyl zinc (DEZ, Zn(Et)₂, Strem Chemicals) was used as the zinc source and demineralized water as the oxygen source for the formation of ZnO. A thermal pre‐treatment of the CA fibers was carried out in the reactor at 195°C for 1 h under vacuum to remove possible residual organic solvents. The VPI process was carried out at 185°C under a constant nitrogen gas flow of 150 standard cubic centimeters per minute (sccm). To ensure the infiltration of ZnO into the CA fibers, they were exposed to DEZ and H₂O vapors for a defined period of time (exposure time) before purging to allow the diffusion of the precursor into the polymer. One VPI cycle consisted of a sequence of 1.5 s dose/90 s exposure/360 s purge for DEZ and the identical sequence for H₂O.

The VPI process was performed at the glass transition temperature (Tg) of cellulose acetate, which is 185°C. It has been previously observed that working at temperatures close to the polymer Tg can facilitate the diffusion of the precursors into the polymer matrix during exposure times due to higher mobility of polymer chains [34]. The Tg of the CA fibers was determined based on thermogravimetric measurements, following the procedure described in our previous study [28].

5.3. Oxygen Plasma Treatment

For the subsequent plasma treatment, 1 cm diameter discs with a thickness of approximately 0.59 mm were pressed from the sample using a tablet press. Disc were used instead of the as‐spun fibrous form to ensure uniform exposure to the plasma across the sample surface and to facilitate reproducible treatment conditions, while still preserving the fibrous structure of the material. They were treated in low‐pressure oxygen plasma of the radio frequency discharge (13.56 MHz) at 100 W for 20 min on both sides. A detailed description of the plasma chamber is in previous publication [94]. The pressure during the plasma treatment was maintained at 10 Pa. Although we did not measure the temperature of the CA fiber discs during the plasma treatment, we can safely state that it was maintained below the CA glass transition temperature (Tg), as similar plasma treatment does not melt highly temperature‐sensitive (melting point of 60°C) polycaprolactone fibers [95, 96].

Additionally, reference planar samples of spin‐coated CA films without infiltrated ZnO were prepared to assess the effect of plasma treatment on the CA. The CA precursor solution was dropped onto the center of a microscopy glass, which was then spun at 3000 rpm for 60 s using a commercial laboratory spin coater (Best Tools) to achieve a uniform coating. After spin coating, the CA films were dried in a vacuum oven to remove residual solvent. The planar films had an approximate thickness of 4 µm as measured using a Confocal Laser Scanning Microscope (Olympus Lext OLS4100). CA films were plasma‐treated under the same conditions as described above for the fiber discs.

5.4. Characterization Techniques

Morphological analyses of produced CA fibers (with and without infiltrated ZnO) were carried out by SEM (MIRA3‐XMU, Tescan, Czech Republic) at an acceleration voltage of 5 kV. Before SEM analyses, fibers were coated with a gold layer (approx. 20 nm) using a laboratory sputter coater EM ACE600 (Leica, Germany) to facilitate sufficient conductivity for morphological observations.

High‐resolution imaging was performed using a field emission scanning electron microscope (SEM, FEI Verios 460 L) at an acceleration voltage of 2 kV, using SE and BSE detectors. For this high‐resolution SEM, fibers were coated with approx. 15 nm carbon layer using the same sputter coater to provide adequate conductivity for detailed morphological observations. Detailed ZnO coating characterizations were performed with an image‐aberration corrected low voltage electron microscopy (LVEM25, Delong Instruments, Brno, Czech Republic) operating at 25 kV. Prior to imaging, all samples were prepared in methanol and bath‐sonicated for 25 min at room temperature. The dispersions were then directly deposited onto holey carbon support films on a 200 square mesh Cu grid (Pacific Grid‐Tech, Micro to Nano, Netherlands), dried and subsequently imaged. Elemental mapping was performed using a Bruker EDS detector integrated into a LVEM 25 electron microscope in STEM mode, operated at 15 kV accelerating voltage.

EDXRF spectrometry was performed using an Elva X Pro spectrometer (Elvatech, Ukraine) to determine zinc content in the samples. The experimental conditions were as follows: X‐ray tube voltage of 45 kV, tube current of 260 µA, excitation beam filters comprising 300 µm nickel and 300 µm aluminum, a 1 mm diameter collimator, spectrum acquisition time of 30 s, and measurements conducted under ambient air atmosphere. Mass concentration values (w/w, normalized to 100%) for zinc were calculated using the fundamental parameter model Oxides E207, which is incorporated within the instrument's default software package. Each sample was measured eight times and obtained results were averaged.

XRD was employed to characterize the crystalline structure of the samples. Measurements were performed using a Smart Lab 3 kW diffractometer (Rigaku, Japan) in Bragg–Brentano geometry, with Cu‐Kα radiation (λ = 0.154 nm) generated at 40 kV and 30 mA. Diffraction patterns were recorded using a Dtex‐Ultra 1D detector over a 2θ range of 10°–90°, with a step size of 0.01° and a scanning speed of 4°/min.

Raman spectroscopy was used for chemical analysis of the samples. The spectra were acquired using a WITec Alpha 300R spectrometer equipped with a 633 nm laser and a 50× objective lens. To prevent damage to the analyzed material, the laser energy was reduced to 5 mW, with a 10 × 10 s acquisition. The Raman spectra were adjusted in the included Witec suite FIVE software for background adjustment only. Before measurement, the instrument calibration was checked against a silicon standard.

Chemical composition of sample surfaces was analyzed using XPS (Kratos Axis SupraTM). Elemental composition was determined from wide spectra and averaged from 3 locations. Hi‐res analysis was performed from spot size 700 × 400 µm, by resolution 0.1 eV, pass energy 20 and flood gun on (0.45 mA). According to database reference [76] CA polymer peak C 1s – CH₃ was calibrated to 285.4 eV. Zn 2p and O 1s peaks of ZnO nanocrystalline layer were calibrated to carbon residuals to 284.8 eV.

Water contact angle (WCA) measurements were performed to assess the surface wettability of the samples. The WCA was assessed using a Surface Energy Evaluation System device (See System E, Advex Instruments, Czech Republic) with a proprietary image analysis software. A 3 µL droplet of deionized (DI) water was dropped onto the surface and allowed to stabilize for 5 s. After this time, the contact angle of the droplet was recorded. Measurements were performed 3 times for each type of sample.

5.5. Testing of Antibacterial Properties

Antibacterial activity of the fibers was evaluated using Cutibacterium acnes (C. acnes, CCM 3343) and Staphylococcus epidermidis (S. epidermidis, CCM 7221), obtained from the Czech Collection of Microorganisms (Faculty of Science, Masaryk University, Brno, Czech Republic). The methodology followed our previously published protocol [28]. Briefly, bacterial cultures were incubated overnight at 37°C and adjusted in saline to an optical density of OD₆₀₀ = 0.1 AU. Bacterial suspensions were spread onto 5% Columbia blood agar plates, and CA fibers (before and after plasma treatment) were placed on the surface. Plates were incubated at 37°C for 48 h (C. acnes) or 24 h (S. epidermidis) under dark conditions to exclude photocatalytic effects of ZnO. After incubation, inhibition zones were measured from the edge of the sample to the boundary of the inhibition zone using a caliper. Penicillin (for S. epidermidis) and gentamicin (for C. acnes) served as positive controls. All experiments were performed in triplicate to ensure reproducibility.

Antibacterial testing was performed on the same day as the plasma treatment, with an interval of approximately 2 h. The samples used were the same ones that had been plasma‐treated 1 year earlier. During this period, the samples were stored in a dark box to prevent photocatalytic effects induced by light. To evaluate the aging effect, antibacterial tests were repeated 1 year after the initial plasma treatment using fibers with 128 ZnO VPI.

Statistical Analysis of fiber diameters from SEM Images: (1) Pre‐processing, of data—none, (2) Data presentation: mean ± standard deviation (SD), (3) Sample size (n) for each statistical analysis: minimum 30 images for each fiber samples, (4) Statistical methods used to assess significant differences with sufficient details—student distribution (t‐test), (5) Software used for statistical analysis—NANOMEASURE.

For antibacterial testing, results are presented as mean ± standard deviation (SD) from three independent measurements.

For EDXRF analysis, results are presented as mean ± standard deviation (SD) from three independent measurements.

Author Contributions

M.R. contributed to writing the original draft, reviewing, editing, performing visualization, investigation, methodology, validation, and data curation. D.F. contributed to writing, reviewing, editing, methodology, investigation, and validation. P.K.C. contributed to writing, reviewing, editing, performing methodology and investigation. A.K. contributed to writing, reviewing, editing, and also performing methodology and investigation. D.P. contributed to writing, reviewing, editing and also performing methodology, investigation, data curation, and validation. S.A. contributed to writing, reviewing, editing, methodology, and validation. K.C. contributed to writing, reviewing, editing, and also performing investigation (performed initial experiments). M.P. contributed to writing, reviewing, editing, methodology, and also performing investigation. K.B. contributed to writing, reviewing, editing, methodology, and performing investigation. L.Z. contributed to writing, reviewing, editing, supervision, resources, methodology, and conceptualization. Z.H. contributed to writing, reviewing, editing, supervision, validation, and funding acquisition. M.K. contributed to writing, reviewing, editing, supervision, validation, and funding acquisition. J.M.M. contributed to writing, reviewing, editing, conceptualization, funding acquisition, supervision, validation, and visualization.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: smll73207‐sup‐0001‐SuppMat.docx.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.