Conceptualizing antibacterial approach for silk-based implants with electrospun cefiderocol nanofibers and PEO-coated Mg3ZnCa

*Corresponding author: Hariharasudan Natarajan. Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Conceptualizing antibacterial approach for silk-based implants with electrospun cefiderocol nanofibers and PEO-coated Mg3ZnCa Hariharasudan Natarajan 1, *, PatriziaImielski 2, Prakash M Alagundi 1 and Periyasamy Siddhan 1 1 Central Silk Technological Research Institute, Center for product development and diversification, Silk Technical Textiles section, Central Silk Board, CSB Complex, BTM Layout, Madiwala, Bengaluru, India. 2 Technische Universität Berlin, Institute of Materials Science and Technology, Fachgebiet Werkstofftechnik/Chair of Materials Science and Engineering, Strasse des 17. Juni 135, 10623 Berlin, Germany. World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 205–217 Publication history: Received on 03 August 2025; revised on 09 September 2025; accepted on 11 September 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.23.3.0831 Abstract Antibacterial functionality for silk-based implants remains as a critical area that needs improvement. This study presents a novel, stepwise antibacterial concept for infection-resistant implants. First, a Mg3ZnCa (ZX31) alloy was selected for its biodegradable properties and coated on the silk base, which was further enhanced using plasma electrolytic oxidation (PEO) for corrosion resistance. Next, electrospun nanofibers loaded with the cefiderocol were applied to the PEO-coated surface. Finally, bacteriophages were immobilized onto the nanofibers. The structure and antibacterial performance of each step were characterized using clinical analysis including electron microscopy imaging. Antibacterial testing against E. coli using ATCC 25922 demonstrated that phage–antibiotic co-release achieved complete bacterial eradication within one hour, compared to eight hours for cefiderocol-loaded fibers alone. Additionally, magnesium corrosion contributed to sterilization after ten hours. These results show the strong synergistic potential of combining magnesium degradation, antibiotic delivery, and phage therapy to prevent earlystage infections on silk implants, offering a promising route for future clinical applications. Keywords: Fibroin Sheet; Implant;ZX31; PEO; Cefiderocol Nanofibers; Antibacterial 1. Introduction Certain patient groups, especially older adults who commonly undergo specific implant surgeries, have weakened organ function, like liver or kidney. They are more vulnerable to the harmful side effects of systemic antibiotics, which can further restrict therapeutic approaches and complicate infection control. The infections which these groups are facing are multidrug-resistant (MRD) bacteria, which poses a major global health concern, making infections harder to treat and steadily reducing the number of effective treatment choices. This problem in some cases is added to the need of a secondary surgery to remove the implant, which doubles the infection possibility. A novel method presented in this research suggests a strategy for reducing the possibility of secondary implant removal surgeries and infection after surgeries. Silk based implants with magnesium alloys offer significant advantages in biomedical applications due to their biodegradability, biocompatibility. Unlike conventional implants, where the silk-based material gradually dissolves in the body, eliminating the need for surgical removal and reducing long-term complications[1,2]. The stiffness of the proposed material can closely resemble natural tissue and ligaments, thus minimizing stress shielding and promoting better integration with surrounding tissue[2]. Additionally, magnesium ions stimulate bone, ligament and muscle regeneration and create an antibacterial environment, lowering the risk of infections[3,4]. However, its rapid weathering World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 205–217 206 in physiological environments poses a significant limitation, as uncontrolled degradation undermines implant stability before sufficient healing has been achieved[5,6]. To address this issue, surface modification techniques on the metal coating, such as plasma electrolytic oxidation (PEO), can be extensively employed to enhance the corrosion resistance of Mg alloys[7]. This method is very similar to anodizing; the difference is that the high-voltage power supply creates glowing sparks and turns the surface material into a flexible semi-ceramic layer[7–13]. Moreover, in vivo studies can highlight the potential of PEO for bone tissue engineering[14]. However, while PEO provides advantageous corrosion protection, it lacks antibacterial properties, making implants susceptible to bacterial colonization and biofilm formation[15]. Electrospinning has emerged as a versatile method for creating nanofiber coatings that incorporate and gradually release therapeutic agents, including antibiotics[16]. This technique uses electric forces to draw charged polymer solutions into fibers, typically ranging from nanometers to a few micrometers in diameter[17]. Electrospun nanofibers are widely applied in tissue engineering, drug delivery, and filtration membranes[17,18]. Many studies have combined polycaprolactone (PCL) and gelatin (Gt) in electrospun nanofibers, incorporating antibiotics for antibacterial properties[19,20]. Sezeret al. [21] explored PCL/Gt nanofibers with gentamicin for bone tissue engineering. They developed biodegradable, osteoconductive scaffolds with multifunctional capabilities, including antibiotic delivery. Bakhsheshi-Rad et al. [22] loaded electrospun nanofibers with ciprofloxacin and deposited the fibers on the surface Mg-1Ca alloy. Although the electrospun nanofibers exhibited the desired drug release and cell culture properties, the porosity of the electrospun layer exposed the underlying magnesium alloy to the corrosive ambient conditions. The weak corrosion resistance of the substrate combined with gas production during the corrosion process led to a reduction in the number of cultured cells. Moreover, while gentamicin and ciprofloxacin are well-known antibiotics, cefiderocol, used in the current study, is a novel antibiotic with minimal resistance concerns[23,24]. Although antibiotics are the most well-known medicines for combating infections, issues such as antibiotic resistance, MDR infections and their adverse effects on organs have driven scientific research towards exploring bacteriophages (phages) as well. Phages are viruses that target and kill specific bacteria. They offer key advantages, like biofilm penetration, and synergy with antibiotics, making them promising options[25–28]. Bacteriophages have diverse shapes and sizes, but the most well-known and studied ones, such as Myoviridae, Siphoviridae, and Podoviridae, generally have an icosahedral (20-sided) head and a tail. The head, also called the capsid, is typically 50 to 200 nm in diameter. The size of the tails can vary significantly depending on the phage family, between 10 to 200 nm in length, and 5 to 20 nm in thickness[29,30]. Imaging of immobilized phages is an open topic in this field, mainly because of their small size[31,25,32], which is one of the subjects we have tackled in this study. Pirlaret al. [33] demonstrated that cefiderocol and phages exhibit a synergistic effect in inhibiting bacterial growth, meaning that their combined use is more effective than using either agent alone. The current study puts the knowledge of the synergic effect of cefiderocol and phages[33] in a novel applicational method to modify the antibacterial implants. In the current conventional applications, clinically antibacterial implants are limited to implants coated with silver nanoparticles[34,35], which cause toxicity[36]. Experimental coatings using zinc, copper, gold, or antimicrobial polymers are still in research or pre-clinical testing[37]. This study introduces a multifunctional, stepwise strategy for bone tissue implants by integrating PEO-coated Mg3ZnCa (ZX31) with cefiderocol-loaded electrospun nanofibers and phages immobilized on the nanofibers. Applying a PEO coating before nanofiber deposition on ZX31 effectively mitigated corrosion of the magnesium alloy. Additionally, cefiderocol is incorporated into the electrospinning solution to provide controlled antibacterial functionality, actively combating post-surgical infections. The comprehensive characterization and testing of the coatings, including corrosion performance, drug release profiles, and antibacterial efficacy, highlight the potential of this novel approach to significantly reduce the risk of implant-related infections. This strategy offers a promising solution to one of the most critical challenges in surgery, paving the way for safer and more effective implant technologies. 2. Materials and methods 2.1. Forming silk sheets Degumming of the mulberry silk cocoons is the first and curial step that involved boiling the cocoons with sodium carbonate to remove the sericin. The process is carried out for 45 minutes, followed by a sample residual degumming process, conducted to confirm the sericin removal is complete. The obtained fibroin is dissolved to form an aqueous solution using a concentrated salt solution of calcium chloride, ethanol and water. The salt solution is then dialyzed using a semipermeable membrane to remove the salt and other impurities leaving a purified, concentrated silk fibroin aqueous solution. The liquid fibroin solution is poured into a mold, subsequently frozen and then freeze-dried to create World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 205–217 207 a thin, porous, sponge-like sheet. A crucial step for biomedical applications is to change the secondary structure of the fibroin to β-sheet structure to make it stable and insoluble by immersion in alcohol that quickly induces the β-sheet formation. 2.2. The alloy for coating silk sheets An ZX31 alloy solution was prepared under argon shielding gas at Technical university of Kaiserslautern, Germany. To check the chemical composition, three samples were taken from the various sections of the solution, solidified, powdered and analyzed using inductively coupled plasma (ICP) spectroscopy (Varian VISTA-PRO, Inc., Palo Alto, California, USA). The ICP results showed an average chemical composition of 95.85 ± 0.6 wt.-% magnesium, 3.12 ± 0.34 wt.-% zinc, and 1.07 ± 0.21 wt.% calcium. The said ZX31 alloy explored in this study is identical to those detailed in various literatures[38]. Further, the silk sheets formed were immersed in the alloy solution under argon shielding gas for effective coating and freeze dried. 2.3. PEO coating To conduct the PEO process, a solution was prepared containing 10 g of Na₃PO₄·12H₂O (M = 380.13 g/mol, Merck, Alfa Aesar, Karlsruhe, Germany), 9 g of Na₂SiO₃·5H₂O (M = 212.14 g/mol, Sigma‒Aldrich, Alfa Aesar, Kandel, Germany), and 1g of KOH (M = 56.11 g/mol, Merck, Saarbrücken, Germany) per liter. The electrical parameters were set as rectangular cathodic pulse mode for 300 s, and a voltage of 200 V by a power supply (Munk Nederland B.V., D400 G500/40 WRGTFW, Kom., Nr.:246241, Hamm, Germany), and the software Visual Plating Controller (Munk Nederland B.V., Oirschot, Germany). A cooling system surrounded the stainless-steel bowl as the cathode, circulating cold water to keep the temperature of the electrolyte constant at ~ 25°C. The abbreviation used for referring to the PEO-coated ZX31 samples in this study is ZX31PEO. 2.4. Potentiodynamic polarization tests To assess the biocorrosion properties of the ZX31 and ZX31PEO specimens, potentiodynamic polarization tests were performed (Ivium, Eindhoven, Netherlands). The square surface of 10 mm2 was exposed to a phosphate-buffered saline (PBS) solution made from 10 OmniPur PBS tablets (Sigma-aldrich, Darmstadt, Germany) dissolved in 1L distilled water, at 25°C (solution composition is 140mM NaCl, 10 mM phosphate buffer, and 3 mM KCl, pH 7.4 at 25°C). To check the repeatability, three samples from each, ZX31 and ZX31PEO groups, were tested. The scan rate was set to 2.5m V/s, covering a range from −200 to 0 mV. Tafel fitting analysis was used to determine the Ecorr and icorr values. 2.5. Electrospinning The electrospinning coating solution comprised Gt type A (Merck, Darmstadt, Germany), PCL (Mw = 80,000 g/mol, Sigma-Aldrich, Darmstadt, Germany), acetic acid (purity ≥ 99%, Merck, Darmstadt, Germany), formic acid (purity≥ 98%, Sigma–Aldrich, Darmstadt, Germany), and cefiderocol (Shionogi, Osaka, Japan). As previously reported [39], the optimal approved concentrations of PCL and Gt, were 10 wt%, and 4 wt%, respectively. The employed concentrations of cefiderocol in this research are 6, 12, and 24 wt%, respectively. To prepare this solution, formic acid was mixed with acetic acid at a 2:1 ratio. PCL was stirred in the acid mixture for 1 h. Subsequently, Gt was added, and the solution was stirred for 30 minutes. Finally, different amounts of cefiderocol were added and stirred until the solution became transparent, indicating full dissolution. The electrospinning machine (FUG Bipolare high-voltage power supplies HCB, AIP Wild, Oberglatt, Switzerland) was set to a voltage of 20 kV, with a 10 cm distance between the collector and the syringe tip, and a flow rate (Syringe pump: TSE Systems 60 Series, Thuringia, East Germany) of 0.1 mL/h. The humidity was maintained below 40%. Since the collector in electrospinning setup should be conductive and PEO coated samples are not so, the PEO-coated samples were affixed to an aluminum foil on the collector using double-sided tape. So, the electrospinning machine recognized the aluminum foil as the collector and the PEO coated samples were covered by nanofibers supposed to be synthesized on the aluminum foil. Synthesizing duration was 2 hours for all samples. The abbreviations used for referring to the ZX31PEO samples covered by 6, 12, and 24 wt% cefiderocol-laden nanofibers in this study are CFD6, CFD12, and CFD24, respectively. 2.6. Scanning electron microscopy (SEM) A Quanta FEG 400 (FEI Company, Hillsboro, Oregon, USA) was used for SEM of the PEO coating and nanofibers before phage incubation. The specimens were gold sputtered for 20 s under an argon atmosphere at 10 Pa and 20 mA. The SEM parameters were set to high vacuum mode with secondary electron detection at 20.00 kV and a working distance of 12.2 mm. A cross-section of the ZX31PEO sample was made after embedding. The diameter of the nanofibers on the top surface was quantitatively analyzed over 100 random measurements of the thickness of the nanofibers via ImageJ (ImageJ software, National Institute of Health)40. World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 205–217 208 2.7. Fourier transform infrared (FTIR) spectroscopic For recognition of the functional groups in electrospun nanofibers, the layer of CFD 12 nanofibers, as the representative sample, was separated and explored by FTIR. Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) was performed on a Bruker Vertex 70 device. (Bruker Optics GmbH & Co. KG, Ettlingen, Germany), covering 4000–400 cm⁻¹. 2.8. Proton nuclear magnetic resonance (¹H-NMR) To evaluate the possibility of hydrogen bonding, proton nuclear magnetic resonance (¹H-NMR) spectroscopy was performed on a Bruker Advance instrument (400 MHz) (Billerica, Massachusetts, USA) on CFD 6, CFD 12, and CFD 24 nanofibers layers, using deuterated solvents (Deutero GmbH, Kastellaun, Germany). Chemical shifts are referenced to residual impurities in the solvents. 2.9. Sessile drop method The contact angle, made by a drop of water and explored surfaces of ZX31PEO, CFD 6, CFD 12, CFD 24 samples, was measured via the sessile drop technique with a digital microscope (KEYENCE VHX-500, Keyence Deutschland GmbH, Neu-Isenburg, Germany) at room temperature. A 1-mL glass micro syringe filled with distilled water was used to dispense a 1.5 µL droplet onto the sample surface, with the piston controlled by a micrometer screw gauge. Each surface was tested five times, and the average contact angle was calculated. ImageJ software was used for analysis, and calculations were based on the model developed by Owens, Wendt, Rabel, and Kaelble[41]. 2.10. Disc diffusion test The disc diffusion method was used to assess the antimicrobial activity of the bare alloy, ZX31PEO, and CFD 6, CFD 12, and CFD 24 samples based on the Clinical and Laboratory Standards Institute guideline[42]. E. coli ATCC 25922 was chosen as the test organism, with cefiderocol serving as the control. The tests were repeated three times. 2.11. Immobilization of the phages Phage CUB-EC was utilized in this study as it has been shown to be specifically effective against E. coli ATCC 25922 (Labor Berlin – CharitéVivantes Services GmbH, Berlin, Germany). It was isolated by the enrichment method according to the process described in literatures [43]. For incubating phages onto the CFD6, CFD12, and CFD24 samples, they were exposed to a phage solution at a concentration of 10⁸ Plaque-Forming Units (PFU)/mL for 1 h, and addressed as CFD6+Ø, CFD12+Ø, and CFD24+Ø, respectively in the following. 2.12. Observing phages on the nanofibers via SEM To observe phages on the nanofibers via SEM, the samples were fixed in 2.5% glutaraldehyde (Serva, Heidelberg, Germany) in 0.1 M sodium cacodylate buffer (Serva, Heidelberg, Germany), post-fixed for 2 h with 2% osmium tetroxide (OsO₄), dehydrated through a graded ethanol series, and air-dried using hexamethyldisilazane (HMDS; Electron Microscopy Science, Hatfield, USA). The samples were carefully mounted on metal stubs. Prior to SEM analysis (GeminiSEM 300 with Image SP software, Carl Zeiss, Oberkochen, Germany), the samples were sputter-coated with gold-palladium (Sputter coater MED 020, Balzer, Bingen, Germany) and stored in a vacuum until examination. Images were captured at 7 kV using a secondary electron detector. 2.13. Observing phages on the nanofibers via TEM After incubation with the phage solution, for preparing TEM slices, the samples were fixed in 2.5% glutaraldehyde (Serva, Heidelberg, Germany) in 0.1 M sodium cacodylate buffer (Serva, Heidelberg, Germany), postfixed with 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, USA) and 0.8% potassium ferrocyanide II (Roth, Karlsruhe, Germany) in 0.1 M cacodylate buffer, dehydrated through a graded ethanol series, and embedded in Epon resin (Roth, Karlsruhe, Germany). Ultrathin sections (70 nm) were then cut from the hardened Epon blocks via an ultramicrotome (Leica, Wetzlar, Germany) with a diamond knife (Diatome, Nidau, Switzerland). These sections were collected on pioloform-coated copper grids (Plano, Wetzlar, Germany) and stained with lead citrate (Merck Millipore, Darmstadt, Germany). The slices were observed with a Zeiss Leo 906 transmission electron microscope (TEM; Carl Zeiss, Oberkochen, Germany) equipped with a slow scan 2K CCD camera (TRS, Moorenweis, Germany) at an accelerating voltage of 80 kV. World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 205–217 209 2.14. Drug release test To examine the release of cefiderocol, a calibration curve was initially created via the Beer‒Lambert law with a PBS at pH 7.4 and 37°C. Subsequently, nanofibrous scaffolds of CFD6, CFD12, CFD24, and CFD6+Ø, CFD12+Ø, and CFD24+Ø, samples were separated from the PEO-coated surfaces, and immersed in 20 mL of PBS at 25°C. At predetermined intervals, every 2 h for the first group and every hour for the second group, 2 mL of the solution was removed from each sample for analysis, and 2 mL of fresh PBS was replaced to continue the release investigation. The amount of cefiderocol released into the PBS was monitored by measuring the UV absorbance at the peak wavelength of 260 nm via a UV spectrophotometer (Eppendorf BioPhotometer, Hamburg, Germany). The cumulative release percentage was then calculated. This process was repeated twice for all the samples to check the accuracy of the tests. Both repeats showed the same results. 2.15. Synergic behavior of the released “phages and antibiotic” test To explore the antibacterial properties of CFD6+Ø, CFD12+Ø, and CFD24+Ø, the samples were washed at least six times with a dip-wash method in sterile distilled water to rinse off non-immobilized phages. One mL of bacterial suspension at a concentration of 5 × 10⁵ CFU/mL was added to each well of a 24-well plate. The samples were subsequently placed into the wells. Bacterial counts were determined via colony-forming unit (CFU) counting at 2 h intervals. At each 2 h interval, 10 µL of the content from each well was taken and added to 990 µL of PBS. This mixture was centrifuged and washed three times to remove any remaining antimicrobial agents. After the final wash, 100 µL of the resuspended 1 mL solution was plated on Tryptic Soy Agar (TSA) (Sigma‒Aldrich, Darmstadt, Germany) plates and incubated at 37°C overnight for bacterial growth assessment. The tests were repeated three times. 3. Results and discussion 3.1. Step 1: PEO coating; characterization PEO results in a typical rough and porous surface layer on the ZX31 alloy surface[8] (Figure 1a). The cracks (marked by arrows) are due to shrinkage during solidification. The cross-section through the coated sample (Figure 1b) highlights that the PEO layer consists of two parts: an inner, dense layer (marked in orange) and outer one (marked in purple), containing the pores. Some of these pores seem to be connected by channels, and they reach deep into the outer layer. The dense inner layer is the one important for corrosion protection of the substrate[44]. Beneath the PEO coating, we see the alloy with its typical eutectic microstructure, consisting of a Mg grains, surrounded by Ca2Mg6Zn3 precipitations at the grain boundaries [38]. Figure 1c shows current versus time during the PEO process. As explained by many studies[13,45,46], after the current‒time diagram passes the sharp peak, the PEO process remains in an almost stable range of current. Thus, here in the first 10s, the surface reached the stable plasma level, and the coating was completed during the process time. Figure 1d depicts the potentiodynamic polarization curves of ZX31 and ZX31PEO in PBS, highlighting the corrosion protection effect of the PEO coating in a corrosive environment. The data derived from the polarization curves are summarized in Table 1. The corrosion current density (Icorr) for the ZX31PEO samples was approximately 15.30 μA/cm², whereas for the uncoated samples, it was 31.23 μA/cm². This disparity demonstrates the positive effect of the PEO coating on the corrosion behavior of the samples. The Icorr of the ZX31PEO sample is reduced to 50 % compared to the uncoated sample, and, concurrently, the corrosion resistance of the ZX31 sample is improved up to twofold due to the PEO coating. Accordingly, the corrosion potential (Ecorr), shifted toward a more positive value from -1.66 V for ZX31, to -1.42 V for ZX31PEO. A more positive Ecorr means that the material is less prone to oxidation and dissolution in a corrosive environment. The corrosion rate corroborates the findings. It is reduced by nearly 51% after PEO coating, indicating improved durability of the material. Table 1 Corrosion data of potentiodynamic polarization analysis of ZX31 andZX31PEO samples Corrosion parameter ZX31 ZX31PEO icorr (µA/cm2) 31.23 15.30 Corrosion rate (µm/year) 102.20 50.05 Ecorr (i=0) (V) -1.66 -1.42 World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 205–217 210 SEM images of a) the top surface microstructure, and b) the cross-section view of the ZX31PEO sample, c) current-time diagram of the PEO process, d) polarization test results of ZX31 and ZX31PEO samples. Figure 1 PEO characterization 3.2. Step 2: Electrospun nanofibers on the PEO coating; characterization and application Figure 2a, Figure 2b, and Figure 2c present typical SEM micrographs of the electrospun nanofibers of CFD6, CFD12, and CFD24. The synthesized nanofibers are straight and randomly placed over and next to each other. The thickness of the fibers with an average diameter of 53.05 nm and a standard deviation of 18.45 nm was evaluated as CFD 6 to be the least uniform sample. The CFD 12 shows a better homogeneity in fiber diameter along the fibers (average diameters: 99.31 nm, standard deviations: 64.52). Finally, for CFD24, it can be observed that there are two classes of very thin and very thick nanofibers. CFD 24 fibers are mainly thicker (average diameters: 110.10 nm, standard deviations: 53.15) than those of the CFD6 and CFD12 nanofibers due to higher percentage of CFD. The important point is that the surface of all nanofibers is smooth, of which the sample CFD 24 is the smoothest after CFD 12 and CFD 6, respectively. Figure 2d exhibits the FTIR spectra of the CFD12 nanofibers as the representative nanofiber. The FTIR diagram shows bonds related to PCL and Gt groups at 2932 cm⁻¹ (asymmetric CH₂ stretching), 2854 cm⁻¹ (symmetric CH₂ stretching) often linked to aliphatic groups, such as the methylene (CH₂) groups in cefiderocol, 1729 cm⁻¹ (carbonyl stretching), 1635 cm⁻¹ (C=O stretching), 1524 cm⁻¹ amide II band, which involves N–H bonding and C–N stretching, 1292 cm⁻¹ (CO and C-C stretching), 1242 cm⁻¹ (asymmetric C-O-C stretching), 1170 cm⁻¹ (symmetric C-O-C stretching), and 724 cm⁻¹ (N-H out-of-plane wagging)[39]. The peak at 1462 cm⁻¹ is often associated with CH₂ bonding vibrations[45]. In PCL and gelatin, the peak at 1363 cm⁻¹ typically corresponds to CH₃ bonding vibrations, which are often seen as symmetric bonding or deformation of methyl groups. It can also be related to the bonding vibrations of -OH groups. For cefiderocol, it corresponds to CH₃ bending or N-O asymmetric stretching in nitro groups. The peak at 1101 cm⁻¹ is commonly associated with C-wagging, and at 1037 cm⁻¹ is usually attributed to C-H in-plane bonding in aromatic compounds[46]. Figure 2e exhibits the 1H-NMR spectrum. 1H-NMR titration was conducted to investigate the hydrogen bonding possibility between PCL/Gt and cefiderocol in synthesized nanofibers of CFD6, CFD12, and CFD24. Initially, the proton signals, in the range of 7 to 10 ppm, appeared broad and unclear. To address these challenges, 1H-NMR spectra were recorded using a methanol-d4: acetic acid-d4 (1:1) solvent mixture. The dominant signals observed (3.31 ppm, 4.79 ppm, and 9.26 ppm) corresponded to the solvent peaks. Thus, the observed broad peak from 7 to 10 ppm mainly is the peak of solvents. The signals within the 1–5 ppm range are related to protons from both Gt and PCL[47,48]. A specific signal at 2.9 ppm, corresponding to the lysine amino acid group in Gt, which is critical for hydrogen bonding, was identified. If increasing the weight percentage of cefiderocol had resulted in more hydrogen bonds, a corresponding chemical shift World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 205–217 211 would be expected at 2.9 ppm. Here, no significant chemical shifts were observed for this signal across the different drug concentrations. The absence of shifts in the 1H-NMR spectra for the three cefiderocol concentrations shows a lack of interaction between the drug and the nanofiber scaffolds. This is important in discussing the drug release properties of these nanofibers which will be investigated later in this article. Figure 2f presents the contact angle measurements of the ZX31PEO sample in comparison with those with nanofibers coating. The nanofiber layer in CFD6 enhances the surface hydrophilicity relative to that of ZX31PEO. However, as the cefiderocol concentration increases in CFD12 and CFD24, the surface becomes more hydrophobic than that in CFD6, indicating a correlation between the cefiderocol content and hydrophobicity. a-c) SEM images showing the morphology a) of CFD 6, b) CFD 12, and c) CFD 24 electrospun nanofibers; d) FTIR spectrum of CFD 12 electrospun nanofibers; e) ¹H-NMR spectra of CFD 6, CFD 12, and CFD 24 electrospun nanofibers in a methanol-d₄/acetic acid-d₄ (1:1) mixture; f) contact angle test images and quantification of it of ZX31PEO, CFD 6, CFD 12, and CFD 24 samples. Figure 2 Nanofibers on top of the PEO coating characterizaltion 3.3. Disk diffusion test Figure 3 presents the antibacterial disk diffusion test results of the bare alloy, ZX31PEO, CFD6, CFD12, and CFD24 samples (left) compared with those of the control sample (right) against E. coli ATCC 25922. The transparent-clear areas are the zones in which the bacteria could not grow. The bare ZX31 sample exhibited greater antibacterial properties than did the ZX31PEO sample, as indicated by the larger transparent inhibition zone around it (marked by a yellow dashed line). An exact disk diffusion diameter cannot be reported for the bare and ZX31PEO samples because bacterial inhibition in these cases does not result from the homogeneous release of an antibacterial agent but rather from patterns caused by chemical reactions. The increased corrosion resistance of the PEO-coated sample, compared with that of the bare alloy, affects its antibacterial effect. ZX31 underwent rapid corrosion, which contributed to bacterial killing, whereas the PEO coating on ZX31PEO provided increased corrosion resistance, thus reducing the bacterial inhibition by corrosion effects. The samples coated with antibiotic-laden nanofibers exhibited varying antibacterial effects depending on the cefiderocol content. With increasing CFD content, the disk diffusion diameters increase, with 26, 28, and 30 mm for CFD6, CFD12, and CFD24, respectively. Surprisingly, these values are still smaller than the diameter of 35 mm measured for the control sample. Even when an antibacterial agent is incorporated into a material, its controlled release is crucial, especially for potential applications in surgeries. One of the key achievements of this research is the successful release of antibiotics. World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 205–217 212 Antibacterial disk diffusion test results of ZX31, ZX31PEO, CFD 6, CFD 12, CFD 24, and control sample against E. coli. Figure 3 Disk diffusion test of the samples with and without nanofibers 3.4. Step 3: Immobilized phages on nanofibers; characterization and application Figure 4a exhibits the SEM image of CFD12+Ø, as the representative of nanofibers incubated with phages. The sample prepared for SEM imaging was not washed so that the immobilized phages (marked in the blue square) and nonimmobilized phages (depicted in a yellow circle) could be captured. The size of the non-immobilized phage is approximately 200 nm. The image indicates that this phage belongs to the Myoviridae family of phages[49,50]. The blue square marks immobilized phages as small dots, similar to all the dots along on the nanofibers in this figure. A comparison of this figure with Figure 2a, Figure 2b, and Figure 2c, at the same magnification, clearly indicates that the small dots are a large number of phages placed on the surface of the nanofibers. The difference between the size of the phage in the yellow circle and the size of the dots as the capsid of the phages is the distance between the nonimmobilized phage and the immobilized phage. a)SEM image of phages on electrospun nanofibers of CFD 12+Ø, b) TEM image of cross-section of CFD 12+Ø. Figure 4 Phages over nanofibers, SEM and TEM look Figure 4b shows a cross-sectional slice through a CFD12+Ø sample, observed via TEM. The white area represents the cross-section of the nanofiber. The dark big gray area and small black areas in the background of it, pointed by arrows, are the pores in the front and at the depth of the picture caused by random placement of nanofibers. The small light circles in the dark gray areas are the phages attached to the fibers, as they were observed as small dots in the SEM images. The yellow arrows in Figure 4b point to a phage at the capsid and tail parts. All the tails could not be captured by TEM because of their thin diameter. However, this image provides information regarding the intact immobilization World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 205–217 213 of the phages without separation of capsids from the tails on the pores between the nanofibers. The release ability and antibacterial performance of the phages are important questions to explore next in this study. 3.5. Drug releasing test Figure 5 presents the results of the drug release tests in PBS for two groups of samples: CFD6, CFD12, and CFD24, containing only antibiotics, and CFD6+Ø, CFD12+Ø, and CFD24+Ø, containing antibiotics and phages. For both groups with and without phages, the release curves cover each other, indicating that the release occurred regardless of the increase in the cefiderocol concentration. The release of the CFD6 was slightly sooner in the first few hours, as the CFD6 curve shows a shift to the left. However, its curve covers CFD12 and CFD24 from the 8th hour. Hydrogen bonding is the most important factor contributing to crosslinking interactions between molecules in an electrospinning solution. However, the 1H-NMR results shown in Figure 2e proved that increasing the cefiderocol weight percentage does not increase the number of hydrogen bonds. El-Lababidiet al. [23] considered the pyrrolidinium group in the cefiderocol structure as the prevention recognition reason. Another significant observation in Figure 5 is the difference between the release in the group with phages and the group without phages, such that the group with incubated phages shows a higher release rate. This means that phages accelerate the release. Cumulative drug release test results of the nanofibers of samples CFD 6, CFD 12, and CFD 24, and CFD6+Ø, CFD12+Ø, CFD24+Ø in PBS. Figure 5 Phages release VS drug release 3.6. Synergic behavior test of phages and antibiotic release Figure 6 exhibits the antibacterial performance of the CFD6+Ø, CFD12+Ø, and CFD24+Ø samples in contact with E. coli bacteria at 2, 4, 6, 8, 10, and 24 h. The bar diagrams show the survival of bacteria as CFUs. The empty space indicates no bacterial growth. The phage group, represented by Ø, shows the growth of bacteria treated only with phages over time. The ZX31PEO group exhibited the growth of bacteria during the mentioned time slots in contact with the ZX31PEO sample. The growth control group, shown by GC, which was not treated with antibiotics, phages, or ZX31PEO, presented a steady increase in CFUs over time, reflecting bacterial growth under normal conditions. During the first 2 h, the CFD6+Ø, CFD12+Ø, and CFD24+Ø samples fully killed the bacteria. Although the CFD6, CFD12, and CFD24 samples exhibited bacterial growth during this period, the CFD6, CFD12, and CFD24 samples presented high, medium, and low bacterial growth, respectively, compared with each other. During the short period of 2 h, two factors could explain this decrease in the bacterial growth: start of phage release and the thickness of the electrospun nanofibers. As depicted in Figure 2, the thickness of the nanofibers increased with increasing amounts of cefiderocol. Therefore, CFD24 presented the highest effective surface along the nanofibers for contacting the bacteria. A greater effective surface, including greater amounts of cefiderocol, killed greater amounts of bacteria. This is also the case for CFD12 and CFD6. After the 4th hour, the CFD6+Ø, CFD12+Ø, and CFD24+Ø samples showed no growth, which means that in these groups the bacteria are killed during the first 2 h. The CFD6, CFD12, and CFD24 samples exhibited almost equal growth of bacteria, meaning that the disposal of the nanofibers has started during this period, and drug release has started more effectively than the effective surface contact of bacteria with the nanofibers. However, the amount of the released drug is still not enough to completely kill bacteria, but compared to the growth control bar, the killing of bacteria has progressed.