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19 February 2026

19 Pages

Bacterial Nanocellulose Wound Dressings with Gentamicin-Loaded Chitosan Nanoparticles for Surgical Site Infection Management

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1
Institute of Pharmaceutical Technology and Center of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Ss. Cyril & Methodius University in Skopje, 1000 Skopje, North Macedonia
2
Center for Medical Microbiology, Institute of Public Health, Dzona Dzeksona bb, 81000 Podgorica, Montenegro
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Faculty of Mechanical Engineering, University of Maribor, Smetanova Ulica 17, 2000 Maribor, Slovenia
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Author to whom correspondence should be addressed.
Polymers2026, 18(4), 510;https://doi.org/10.3390/polym18040510
This article belongs to the Special Issue Advances in Natural Polymers: Cellulose and Lignin
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Abstract

Surgical-site infections (SSIs) represent a significant healthcare burden, often complicating wound healing and recovery. To overcome the limitations of systemic antibiotic administration, such as toxicity and poor localization, this study aimed to develop a bioactive dressing utilizing bacterial nanocellulose (BNC) impregnated with gentamicin-loaded chitosan nanoparticles (GNP). Chitosan nanoparticles were synthesized via ionic gelation with sodium tripolyphosphate (TPP) and optimized using a one-factor-at-a-time (OFAT) approach to control particle size, polydispersity index (PDI) and zeta potential. The optimized nanoparticles were impregnated into BNC disks, and the resulting composite was characterized using FTIR and Raman spectroscopy, XRD and SEM. Antimicrobial efficacy was evaluated against Klebsiella pneumoniae, while biocompatibility was assessed using MTT assays and cell-adhesion studies on human fibroblasts. The optimization process yielded stable, monodisperse nanoparticles with a mean size of 80.07 nm and a PDI of 0.192. SEM imaging confirmed the successful integration of nanoparticles into the BNC nanofibrillar network without compromising the membrane’s structural integrity. The BNC-GNP composite demonstrated significant antimicrobial activity against K. pneumoniae, comparable to free gentamicin solution. Furthermore, in vitro studies revealed good biocompatibility, with cell viability exceeding 70% and sustained fibroblast adhesion, although cell-attachment density decreased with higher nanoparticle concentrations. The developed BNC dressing containing gentamicin-loaded chitosan nanoparticles presents a promising multifunctional biomaterial. It effectively combines local infection control with a biocompatible environment suitable for tissue regeneration, offering a novel approach for the postoperative treatment of surgical-site infections.

1. Introduction

Surgical-site infections (SSIs) remain a persistent healthcare challenge, accounting for a significant proportion of hospital-acquired infections. These infections are primarily mediated by Staphylococcus aureus, Pseudomonas aeruginosa and multidrug-resistant Klebsiella pneumoniae [1,2]. They severely disrupt the wound-healing process and prolong postoperative recovery, leading to a higher risk of complications, as well as a financial burden. The risk of SSIs is particularly heightened in immunocompromised patients and patients undergoing lengthier invasive procedures [3,4].
Current strategies for the prevention of SSIs rely on preoperative antiseptic measures and antibiotic prophylaxis [5,6,7]. Although the systemic administration of antibiotics has been the mainstay option for successfully mitigating the incidence of SSIs, it comes with the risk of potential systemic toxicity and adverse effects, and it often leads to a disrupted gut microbiome, which in turn can facilitate the subsequent infection with C. difficile [8,9]. Furthermore, the antibiotics’ efficacy can be impeded by poor localization at vascular compromised sites and in cases where biofilms are formed [10]. Hence, approaches utilizing local antimicrobial treatment, including biomaterials and medical devices that facilitate the administration of antibiotics, have gained recent interest. The focus is on formulations that are biocompatible, provide improved antibiotic retention and controlled release, while simultaneously supporting tissue regeneration [11,12,13,14,15].
In this direction, various naturally occurring polymers have been studied as both favorable dressing materials and as a part of drug delivery systems. Among them, BNC has been extensively researched, as it offers critical attributes suitable for wound-healing applications, including its biocompatibility, high water-holding capacity and suitable mechanical properties. It forms a physical barrier to microorganisms, while simultaneously allowing gaseous exchange and maintaining a moist environment. Alongside these advantages, it also has a highly porous structure, offering the possibility of impregnation with various therapeutic substances, without suffering any major structural changes that hinder its performance. Its fibrous structure further aids the cell attachment and proliferation, critical segments in the wound-healing process [16,17,18,19].
Chitosan (CS) is another naturally derived polymer, a polysaccharide that demonstrates significant potential in biomedical applications due to its biodegradability, biocompatibility and intrinsic antimicrobial properties. The most plausible mechanism of action against microbes is the interaction with the cell membrane, altering cell permeability, causing cytosolic leakage and inducing lysis. This stems from the amino groups of the polymer chain that can be protonated, giving the molecule a positive charge which allows for electrostatic interactions with bacterial cell membranes [20,21,22,23]. Additionally, CS can stimulate fibroblast proliferation and angiogenesis, further improve the wound healing process and reduce the formation of scar tissue [24]. When formulated into nanoparticles, CS can facilitate the site-specific, controlled release of antibiotics, complement the benefits of BNC and improve the bioavailability of therapeutics in compromised tissues [25]. Incorporating CS nanoparticles loaded with antibiotics into BNC matrices may result in a multifunctional composite biomaterial capable of simultaneously controlling infection, reducing adverse effects and providing a suitable environment for tissue regeneration.
Given these considerations, this study aims to develop a bioactive BNC dressing material, utilizing the newly produced gentamicin-loaded chitosan nanoparticles (GNP) into its 3D nanofibrillar network.
Recent advancements in BNC composites have successfully incorporated antimicrobial agents such as silver nanoparticles, metal–oxide nanoparticles and various organic fillers in order to improve wound-healing outcomes. Although these systems were successful in inhibiting common pathogens, most research focused on general wound care, nerve regeneration or hemostasis, not including the management of postoperative surgical-site infections specifically [26,27,28,29].
Hence, our study aims to address this gap by synergizing BNC and gentamicin-loaded CS-TPP nanoparticles, a combination which, in our knowledge, has not been explored yet. This multifunctional strategy aims to utilize the structural benefits of BNC and the stability provided by the nanoparticles into a composite scaffold, to overcome the common clinical limitations of standard SSI treatments.
The obtained formulations were assessed in terms of physicochemical characteristics and behavior in contact with biological systems, i.e., relevant pathogenic bacteria and cells. The proposed system could offer a novel approach for postoperative wound treatment with the potential to improve patient outcomes by addressing both SSIs prevention and wound healing.

2. Materials and Methods

2.1. Materials

CS (medium Mw, viscosity of 200–800 cP and 75–85% degree of deacetylation) was purchased from Merck Milipore (Burlington, MA, USA; lot number: #SLBG4282V), as well as the tripolyphosphate sodium (TPP) and gentamicin sulfate (GS). For the cell-culture studies, Dulbecco’s Modified Eagle’s medium (DMEM) purchased from Lonza (Walkersville, MD, USA), was used as cell-culture medium, supplemented with 10% Fetal bovine serum (FBS) purchased from Sigma Aldrich (Taufkirchen, Germany) and 1% pen-strep solution (Sartorius, Göttingen, Germany).

2.2. Nanoparticles Preparation and Optimization

Chitosan nanoparticles were synthesized via the ionic gelation technique, utilizing sodium tripolyphosphate (TPP) as a crosslinker. For the gentamicin sulfate-loaded nanoparticles, the antibiotic (5, 10 or 40 mg) was dissolved in the chitosan solution prior to the homogenization process. A TPP aqueous solution, with concentrations ranging from 0.25 to 6.25 mg/mL, was introduced dropwise into the chitosan solution (concentrations of 0.5 or 2.0 mg/mL) under continuous homogenization over an 8 min period. Agitation was maintained throughout the process using magnetic stirring at speeds of 400 or 700 rpm, or by an ultrasonic probe (300VT Ultrasonic Homogenizer, BioLogics, Manassas, VA, USA) operated at 50% power intensity; in specific instances, a combined approach using both magnetic stirring and ultrasonication was employed. To optimize particle size, particle size distribution and zeta potential, the processing parameters (homogenization method) and formulation variables (chitosan-to-TPP mass ratios ranging from 10:1 to 1:5 and gentamicin sulfate content) were systematically varied using a one-factor-at-a-time (OFAT) approach (Table 1).
Table 1. One-factor-at-a-time experiments.

2.3. Particle Size, Particle Size Distribution and Zeta Potential

The mean particle size (Z-average), particle size distribution (polydispersity index, PDI), and zeta potential (mV) of the nanoparticles were determined using dynamic light scattering (DLS) analysis with a Zetasizer Nano Series instrument (Nano-ZS, Malvern Instruments, Worcestershire, UK). Measurements were carried out at 25 °C using clear disposable zeta cells. For both particle size and zeta potential analysis, the refractive index and viscosity of the dispersant were set to 1.330 and 0.8872 cP, respectively. Measurements were recorded over 60 s, with a measurement angle of 175°. Prior to measurement, all samples were diluted 1:10 with distilled water and each measurement was performed in triplicate (n = 3) to ensure reproducibility. Results are reported as mean ± standard deviation.

2.4. Chromatographic Method

The analytical method was carried out as described by Saleh et al., 2022 with slight modifications on RP-C18; 125 × 4 mm; 5 µm column on Agilent 1200 HPLC-UV system with isocratic elution at a constant flow rate of 1.1 mL/min. Separation was achieved on a single ODS (C8) column measuring 15 × 0.45 cm with a particle size of 5 µm. The mobile phase was prepared by mixing methanol, water, and glacial acetic acid mixed in a volumetric ratio of 70:25:5 (v/v), into which sodium 1-heptane sulfonate was incorporated at a concentration of 0.5% w/v. The ion-pairing reagent was added directly to the combined solvent mixture.

Standard and Sample Preparation

Working standard solutions of gentamicin were prepared at concentrations of 1, 0.75, 0.5, 0.25, 0.1, and 0.05 mg/mL. The gentamicin reference standard was dissolved in water, followed by the addition of methanol and o-phthalaldehyde (OPA) reagent, with the final volume adjusted using methanol. These solutions were heated in a water bath at 60 °C for 15 min and cooled to ambient temperature. For nanoparticle samples, a defined volume of the nanoparticle dispersion underwent ultrafiltration through Vivaspin 20 tubes (Sartorius, Göttingen, Germany), and the filtrate was derivatized following the identical procedure applied to the standards. For gentamicin sulfate injection samples, 2 mL (equivalent to 80 mg gentamicin) was diluted to 100 mL with water; 2.5 mL of this dilution was further diluted to 100 mL with water, and 10 mL of the resultant solution was transferred to a 25 mL volumetric flask containing 5 mL methanol and 4 mL OPA reagent, with final adjustment to volume using methanol. The calibration curve was constructed from a 50 mg/mL gentamicin sulfate stock solution in water, subjected to serial dilutions and derivatization with methanol and OPA to achieve final concentrations of 0.8–2.4 µg/mL; peak areas were plotted against concentration for quantification. Prior to HPLC injection, all standard and sample solutions were filtered through a 0.45 µm membrane.

2.5. Preparation and Loading of BNC Films

The BNC films were prepared according to the methodology outlined in the article by Jančič et al. [30]. After purification in 0.5 NaOH the BNC films were cut into circular pieces with a punching tool with a diameter of 16 mm and autoclaved for 20 min at 121 °C and 2.1bar (CertoClav VacPro, CertoClav Sterilizer GmbH, Leonding, Austria). The sterile disks were further used for loading gentamicin sulfate, chitosan nanoparticles, and nanoparticles loaded with gentamicin sulfate. The procedure involved depositing 0.5 mL of dispersion into a 12-well plate, where an 8 mm diameter (surface = 50.24 cm2) BNC disk was placed. The plate was maintained overnight under controlled conditions to allow the liquid to be absorbed into the membrane’s pores. The concentrations of gentamicin sulfate, chitosan nanoparticles, and nanoparticles loaded with gentamicin sulfate are given in Table 2. The loaded and blank nanoparticles were previously prepared in aseptic conditions from sterile filtered solutions, using the previously described method.
Table 2. Code used for each sample with corresponding to the mass of gentamicin and nanoparticles.

Nanoparticles Preparation in Aseptic Conditions

The CS and TPP solutions were filtered through 0.2 μm RC filters into sterile test tubes in a laminar-flow chamber Labculture class II (ESCO, Taichung, Taiwan). The tripolyphosphate solution was added dropwise to the chitosan solution over an 8 min period, with 50% ultrasonic agitation applied. Solutions of gentamicin sulfate at various concentrations, nanoparticles containing gentamicin sulfate at different concentrations, and empty nanoparticles were prepared.

2.6. FTIR and Raman Spectroscopy

Fourier transform infrared (FTIR) spectroscopy was performed using a Cary 630 FTIR spectrometer equipped with a ZnSe engine and Diamond ATR module (Agilent, Waldbronn, Germany). Measurements were conducted at a spectral resolution of 4 cm−1 over the range of 650–4000 cm−1. Raman spectroscopy was carried out on an ATR 3000 portable Raman spectrometer (Optosky, Xiamen, China) with an integration time of 60,000 ms and a detection power of 400 mW. To highlight the difference between samples, the deconvolution process, by Savitzky–Golay first-derivative analysis, was performed.

2.7. SEM

The surface morphology of the BNC-based disks was analyzed using a Carl Zeiss FE-SEM Supra 35 VP (Carl Zeiss NTS GmbH, Oberkochen, Germany). Pieces of BNC-based disks were cut, positioned onto a holder by double carbon tapes and sputter coated with a thin layer of gold. Different magnification images were taken, according to regions of interest and the size of investigated structures, in a high vacuum at 1 kV accelerating voltage using secondary electron mode. The fiber diameter distribution of the BNC disk was determined from SEM images using ImageJ 1.51w software manually measuring randomly selected nanofibers at 100 different points. The orientation of the fibers was determined using the OrientationJ plugin [31] to the ImageJ software 1.51w.

2.8. XRD

The XRD diffractograms of the BNC-based disks were acquired using a D2 X-ray diffractometer (Bruker Siemens, Karlsruhe, Germany). These measurements were performed by utilizing CuKα radiation (λ = 0.15406 nm) at a current of 10 mA and a voltage of 30 kV. Spectra spanning the 10–30° 2θ range were recorded with a step size of 0.03°. The background signal, obtained from an empty Si holder, was subtracted from the sample data to ensure accurate results. The crystalline percentages were determined using Diffrac.Eva software V7, which determines crystallinity by analyzing the ratio of crystalline region’s area to the total area in the XRD spectra.

2.9. In Vitro Studies

2.9.1. Antimicrobial Activity

The analysis was conducted on a Klebsiella pneumoniae strain susceptible to gentamicin. Bacterial identification was performed using the MS-Vitek system. Antimicrobial resistance was evaluated by the disk diffusion method and the automated Vitek2 system with GN 439 and XN 26 cards. Susceptibility testing was carried out on Mueller–Hinton agar (pH 7.2–7.7) (Pro Media, Miami, FL, USA), and result interpretation followed EUCAST 2024 guidelines (The European Committee on Antimicrobial Susceptibility Testing). Klebsiella pneumoniae was isolated from the urine of a patient with a urinary tract infection. The urine sample was initially cultured on blood agar and CPS medium (chromogenic plate agar—bioMérieux, Marcy l’Étoile, France), where ≥100,000 CFU/mL (colony-forming unit) of bacterial growth was detected. The grown colonies were placed into tubes containing 0.5 mL of physiological saline, and turbidity was adjusted to 0.5 McFarland standard (108 CFU/mL) using a turbidimeter. Using a swab and rotor, the prepared suspension was inoculated onto Mueller–Hinton plates with a standard thickness of 4 mm for further testing. For some analyses, disks containing nanoparticles (diameter 10 mm) were used, while for other analyses the agar dilution method (the well-diffusion method) was applied (well diameter 6 mm; well capacity 50 µL). The diameter of the inhibition zones surrounding both nanoparticles’ wells and nanoparticles-impregnated nanocellulose scaffolds were measured with calipers in millimeters after incubation at 35 ± 1 °C for 16–20 h.

2.9.2. Cell Cultures

During the experimental work, a cell line of BJ cells, derived from neonatal male skin, was used. These cells are human fibroblasts, which belong to the group of cells present in connective tissue and are utilized as a model for various biological and physiological studies. For cell maintenance, Dulbecco’s Modified Eagle’s medium (DMEM) was used, supplemented with 10% FBS and 1% antibiotics. The cell cultures were kept in a humidified incubator with 5% CO2 at 37 °C.

2.9.3. Cell Viability Studies

Cell viability was evaluated using the MTT assay, which measures the ability of living cells to convert yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) into purple formazan crystals through mitochondrial enzyme activity. In brief, approximately 5 × 105 fibroblasts per well were seeded into 96-well plates. Samples of BNC-15 G, BNC-15 NP, BNC-15 NP G, and blank (BNC) were placed in 25 mL DMEM and incubated on a shaker at 37 °C, 30 rpm for 24 h. Positive control (0.2% Triton X-100) and negative control (high-density polyethylene) were included. Then, the resulting eluates were filtered through 0.22 μL filters, and dilutions were prepared to achieve concentrations of 10 and 5 G, NP, and NPG. Cell culture medium was replaced with 200 μL of these eluates, followed by 48 h incubation at 37 °C. Subsequently, 20 μL of MTT solution (4 mg/mL in physiological solution) was added for 4 h, after which 200 μL DMSO were added to dissolve the formazan crystals. Absorbance was measured at 595 and 560 nm using a Victor X4 Multilabel Reader (Perkin Elmer, Waltham, MA, USA). Additional dilutions were prepared to ensure measurements within the linear response range.

2.9.4. Cell Adhesion

Disks were pre-hydrated in 3 mL DMEM (1 h, at room temperature) to facilitate adherence. According to the literature, approximately 50,000 cells per disk are needed for a successful experiment. Consequently, 150 μL of the redispersed cells were transferred into a vial, and 1.2 mL of DMEM was added. The cell concentration applied to each disk was 106 cells per μL. Following the initial attachment (40 min incubation), 1 mL DMEM was added to each well, and cultures were maintained for 7 days with regular medium changes. Post-cultivation, the disks were stained with Acridine orange (10 μL per 100 μL medium, 5 min), rinsed with DPBS, and fixed in 10% formalin (1 h). After DPBS washing, samples underwent dehydration through an ascending ethanol gradient (25%, 50%, 75%, 100%; 2–3 min per concentration). Disks were future depicted by SEM microscopy, using the same set up as described under Section 2.7.

3. Results and Discussion

To evaluate how formulation and processing parameters affected the characteristics of the chitosan nanoparticles, three sets of formulations were prepared by varying the homogenization method, chitosan-to-TPP ratio, and gentamicin sulfate concentration using a one-factor-at-a-time (OFAT) approach. The resulting nanoparticles were characterized for their physicochemical properties, including mean particle size, PDI, zeta potential, as well as encapsulation efficiency.

3.1. Physicochemical Characterization of the Prepared Nanoparticles

3.1.1. Particle Size, PDI, Zeta Potential and Encapsulation Efficacy

Particle size and polydispersity index (PDI) measurements were conducted for all formulations (Supplementary Figure S1), with results expressed as mean ± standard deviation (SD) (Figure 1).
Figure 1. (A) Particle size distribution of the gentamicin-loaded formulations (NP-C-1 to NP-C-3), each measured three times (represented by the different graph colours); (B) Combined graph representing the average values and the corresponding standard deviation values (SD) for Z-average (d.nm, left y-axis) and polydispersity index (PDI, right y-axis) for all formulations.
The initial optimization focused on homogenization techniques. Comparative analysis (Figure 1A,B) revealed that ultrasonic homogenization (50% power, 8 min duration) produced nanoparticles (NP-B-3) with the most monodisperse size distribution (PDI = 0.433 ± 0.001), outperforming the methods using a magnetic stirrer, which is in accordance with the existing literature [32,33,34,35,36]. Briefly, the cavitation caused by ultrasonic homogenization produces localized “hot spots” with extreme temperatures and pressures, as well as intense mechanical shear forces. These forces are highly efficient at breaking down microaggregates and ensuring uniform TPP distribution within the CS solution at the moment of gelation. In contrast, magnetic stirring relies on macroscopic fluid flow to distribute the crosslinker, which often results in localized regions of high TPP concentration, leading to inhomogeneous crosslinking density and broader particle size distribution.
Following method standardization, subsequent optimization examined chitosan-to-TPP mass ratios. While formulations prepared with 0.5 mg/mL chitosan and 6.25 mg/mL TPP (NP-F) had the lowest values for particle size, the combination of 2 mg/mL chitosan and 2.5 mg/mL TPP (NP-C) was chosen for further development since it demonstrated better PDI. At molecular level, the Z-average size is dictated by the degree of crosslinking density. As the content of TPP increases, a greater number of NH3+ groups on the chitosan chains become neutralized and linked by P3O105− ions, which pulls the polymeric chains closer together and shrinks the hydrodynamic volume of the particle. However, when the TPP concentration exceeds the optimal threshold, it leads to complete neutralization of the surface charges of the developing nanoparticles. The elimination of the electrostatic repulsion then allows the attractive van der Waals forces to dominate, causing the particles to overcome the potential energy barrier and undergo aggregation or flocculation. This results in broader particle size distribution, which explains why the PDI initially improves as TPP at higher concentrations is added, but degrades if an excess is used [37,38,39].
Final optimization addressed gentamicin sulfate incorporation, revealing a direct correlation between antibiotic concentration and particle size. As it is reasonable to expect, higher concentrations of gentamicin sulfate resulted in an increase in the particle size, since the drug incorporation into the chitosan–TPP matrix physically displaces space, causing swelling. Additionally, gentamicin interacts electrostatically with the polymeric network, promoting matrix expansion [40]. The encapsulation efficacy for all formulations was high (75–88%), with a gradual increase as the gentamycin/chitosan ratio decreased.
Since the lowest values for particle size and PDI were measured for the lowest amount of gentamicin sulfate (NP-C-1), this formulation was chosen as optimal and used for the nanocellulose disks’ impregnation. The measured zeta potential (21.311 ± 1.747 mV) further underlines its stability [41].

3.1.2. FTIR and Raman Spectroscopy

The optimal nanoparticles formulation (NP-C-1), as well as the BNC disks impregnated with varying concentrations of gentamicin sulfate solution, empty nanoparticles and gentamicin-loaded nanoparticles were subjected to FTIR and Raman analysis. The spectra of their pure components were obtained as well (Figure 2).
Figure 2. FTIR spectra of (A) empty nanoparticles (NPs), gentamicin-loaded nanoparticles (GNPs) and their individual components; (B) empty BNC, BNC impregnated with varying concentrations of gentamicin sulfate solution, empty nanoparticles and gentamicin-loaded nanoparticles; Raman spectra of (C) empty nanoparticles (NPs), gentamicin-loaded nanoparticles (GNPs) and their individual components; (D) empty BNC, BNC impregnated with varying concentrations of gentamicin sulfate solution, empty nanoparticles and gentamicin-loaded nanoparticles.
From the provided FTIR spectra in Figure 2A, several structural interactions between chitosan and TPP within the nanoparticles can be noted. Firstly, distinct P=O (1204 cm−1) and P–O–P (868 cm−1) stretching bands can be observed, confirming the ionic interactions between the polyphosphate groups and chitosan’s positively charged primary amines. Furthermore, a shift in the chitosan amide I from 1653 cm−1 to 1706 cm−1 is visible, indicating the formation of strong ionic crosslinks that stiffen the C=O bond or disrupt the native hydrogen bonding network that typically lowers the vibrational frequency in the bulk polymer. This tightening of the molecular structure points towards successful ionotropic gelation and indicates the formation of stable nanoparticle matrix. TPP-associated bands at 1209 cm−1, 1100 cm−1, and 890 cm−1 are also present, with a shift in the P–O–P bridge vibration, likely reflecting additional electrostatic interactions with chitosan’s amino groups [42].
The N-H bending vibrations characteristic of gentamicin’s primary aromatic amines, which are typically found at 1614 cm−1 and 1522 cm−1, are detected in the spectrum of the drug-loaded nanoparticles. While the intensity of these bands is relatively low—a consequence of the relatively low drug-to-polymer ratio and the high encapsulation efficiency, their presence is distinct from the signal of the empty nanoparticles. The bands are noted to be highly overlapped with the broadened amide bands of the chitosan matrix, resulting in a broadened spectral envelope in the 1500–1650 cm−1 region. The successful identification of these specific amine signals, despite the significant matrix interference, confirms that gentamicin is chemically and physically retained within the nanoparticles [43]. In addition, similar changes can be observed in the Raman spectra (Figure 2C), where the presence of chitosan is further noted in the nanoparticles’ spectra, since a double-band with maximums at 922 and 888 cm−1 is assigned to vibrations in the chitosan molecule. In addition to these changes, in the gentamicin-loaded nanoparticles’ spectra distinct bands from gentamicin sulfate are present at 1350 and 1167 cm−1, confirming its successful entrapment in the chitosan–TPP matrix. In these spectra the amid I band from chitosan is absent since the higher-intensity vibrations from gentamicin overlap in this region [44].
The FTIR spectra presented in Figure 2B are mainly characterized by bands originating from nanocellulose, especially the strong C–O–C and C–O stretching bands observed between 1000 and 1200 cm−1, which correspond to glycosidic linkages [45,46,47]. Due to the comparatively low concentrations of chitosan nanoparticles and gentamicin sulfate relative to nanocellulose, and the potential for band overlaps, their spectral contributions are largely concealed, which can also be observed in the obtained Raman spectra of the samples (Figure 2D). Although subtle broadening and increased intensity in the amide I (C=O stretching, ~1640–1650 cm−1) and amide II (N–H bending, ~1550 cm−1) regions can be seen in NC samples impregnated with nanoparticles, possibly reflecting the presence of chitosan’s amine and amide groups, these changes are still insufficient to provide satisfactory information on the structural and chemical changes in the sample [48]. Deconvoluted spectra (Supplementary Figure S2) demonstrate that CS and TPP bends, dominant in the 1350–1700 cm−1 region are highly concealed in the NPs-impregnated BNC samples. The composites spectra highly resemble the blank BNC as well, showcasing the reason why no difference between samples can be observed.

3.2. Morphology Assessment

The macroscopic image of the dried BNC disk (Figure 3) revealed a homogeneous, semi-transparent appearance, typical for dried BNC material. The surface was predominantly smooth but showed gentle wrinkling due to the drying process. No visual differences were observed among the disks; therefore, only one macroscopic image of a representative disk is included. On the other hand, SEM imaging revealed the morphological changes in nanocellulose disks impregnated with varying concentrations of gentamicin sulfate solution, chitosan nanoparticles without gentamicin sulfate and chitosan nanoparticles with gentamicin sulfate (Figure 3). The control sample (BNC) exhibited a typical interwoven, fibrillated nanocellulose structure with a uniform surface texture, nanometer-size pores, and fibers ranging from 5 to 45 nm in width, with an average fiber diameter of 24.3 ± 7 nm. The addition of gentamicin sulfate retained the fibrillated structure but affected the fiber width and size of the pores formed, resulting in decreased porosity, higher average fiber diameter and a denser surface layer with increasing concentrations of gentamicin sulfate. The densest surface layer was observed at the highest concentration (BNC-15GS-Sol) with an average fiber diameter of 56.1 ± 23 nm and a relatively wide diameter distribution up to 140 nm. Samples containing empty nanoparticles (BNC-NP) showed a denser, opaque surface with visible spherical nanoparticles and reduced fibrillated structure visibility. As the concentration of nanoparticles increased, the level of agglomeration also increased, covering the cellulose surface. In the samples impregnated with gentamicin-loaded nanoparticles (BNC-GNP), the surface structure becomes even denser, revealing visible nanoparticles and agglomerates. This density increases proportionally with the concentration of the nanoparticles, which parallels the behavior observed in BNC samples containing empty nanoparticles.
Figure 3. Macroscopic image of BNC disk (scale bar corresponds to 5 mm) and SEM micrographs of BNC impregnated with varying concentrations of gentamicin sulfate solution (BNC-5GS-Sol, BNC-10GS-Sol and BNC-15GS-Sol), with empty (BNC-5NP, BNC-10NP and BNC-15 NP) and gentamicin-loaded nanoparticles (BNC-5GNP, BNC-10GNP and BNC-15 GNP) with included color-coded images, where different colors represent various directions of nanofibers, following the half-circle color scheme depicted on the right side of the BNC micrograph. The scale bar in the SEM and color-coded images correspond to 200 nm. The BNC, BNC-5GS-Sol, BNC-10GS-Sol and BNC-15GS-Sol histograms illustrate the distribution of the nanofiber diameters, along with the calculated average diameter (DA ± SD).
Additionally, the orientation of the BNC-based disks was assessed utilizing the OrientationJ plugin for ImageJ, as presented in the inserts of Figure 3. The color-coded HSB images elucidated the preferential fiber orientation in degrees, where hue represents the fiber alignment in accordance with the half-circle color scheme illustrated in the BNC micrograph, and saturation indicates the degree of orientation coherency [49]. The predominance of a single color region is not observable in any of the samples, indicating an overall network with fibers oriented in multiple directions without preferred orientation. Particularly, control sample and BNC disks impregnated with various concentrations of gentamicin sulfate solution exhibit clearly visible fibers with multicolored regions, including green (−45°), pink (+75°), blue (+35°) and purple (+50°) hues. Visibility of individual nanofibers, and consequently orientation, diminishes with the deposition of empty or gentamicin-loaded nanoparticles on BNC disks due to speckled areas, which become even larger as the concentration of empty nanoparticles increases. This is even more pronounced in the presence of gentamicin-loaded nanoparticles. However, it is evident that at the highest concentrations of empty nanoparticles and with gentamicin-loaded nanoparticles, the images exhibit a higher orientation coherency, indicating a more homogeneous distribution of the components. Specifically, in the case of BNC-15NP, the green color (−45°) is most prominent, while in BNC-15GNP, both green and pink (+75°) hues are dominant.

3.3. XRD

The crystallographic structure of the BNC disks impregnated with gentamicin sulfate solution, empty nanoparticles, and with gentamicin-loaded nanoparticles was analyzed using X-ray diffraction spectroscopy. The XRD patterns in Figure 4A revealed a semicrystalline nature with four crystalline regions and a broad amorphous region within the 10–30° range. Diffraction peaks of pure BNC were observed at 14.7°, 16.9°, 20.7° and 22.8° 2θ angles, with two main peaks located at 14.7° and 22.8° 2θ angles, corresponding to the (100), (1–10), (110) and (200) crystal planes of the elementary cellulose crystalline structure, specifically the triclinic Iα and monoclinic Iβ phases [50,51,52]. The XRD patterns of BNC loaded with GS, NP, and GNP exhibit distinct peaks characteristic of the cellulose I crystal without any additional peak, confirming that all samples preserve their fundamental nanocellulose crystalline structure. The crystallinity of pure BNC was 60.9%, while impregnations of BNC with gentamicin sulfate solution, empty nanoparticles or with gentamicin-loaded nanoparticles slightly increased the crystallinity up to a maximum value of 66.8% (Figure 4B) but does not change the basic structure. Lower concentrations of GS-Sol, NP or GNP generally show better structural organization, and no separate crystalline phases appear, indicating good integration of the components [53].
Figure 4. (A) XRD patterns and (B) crystallinity of BNC disks impregnated with gentamicin sulfate solution (BNC-GS-Sol), impregnated with empty (BNC-NP), and gentamicin-loaded nanoparticles (BNC-GNP). The Figure includes pure BNC as a control.

3.4. Antimicrobial Activity

Klebsiella pneumoniae was selected as a representative Gram-negative pathogen for the initial antimicrobial evaluation due to its well-documented involvement in hospital-acquired and surgical-site infections, its pronounced capacity for biofilm formation, and its increasing antimicrobial resistance, including extended-spectrum β-lactamase (ESBL) and carbapenem-resistant strains, which make it a clinically relevant and challenging test organism [54].
The antimicrobial activity test was conducted on the gentamicin-loaded nanoparticles (GNP) and the BNC disks impregnated with gentamicin-loaded nanoparticles (BNC-GNP), with empty nanoparticles (NP), bacterial nanocellulose without NPs (BNC) and bacterial nanocellulose impregnated with empty NPs (BNC-NP) as controls. Gentamicin sulfate solution (GS solution) and BNC disks with gentamicin solution (BNC-GS-Sol) were also tested for comparison (Figure 5). Results were calculated as an area of an annulus, subtracting the diameter of the well or BNC disk, accordingly, and are presented as a mean value ± SD (Figure 5C).
Figure 5. Antimicrobial activity testing on (A) empty chitosan nanoparticles (NP), dispersions of gentamicin-loaded nanoparticles (GNP), gentamicin solution (GS-Sol), bacterial nanocellulose membranes with empty nanoparticles (BNC-NP), bacterial nanocellulose membrane with incorporated nanoparticles containing gentamicin (BNC-GNP), bacterial nanocellulose membranes with gentamicin solution (BNC-GS-Sol), all containing 5 μg (first row), 10 μg (second row) or 15 μg (third tow) gentamicin per sample, excluding empty (NP) samples; (B) Empty bacterial nanocellulose membrane (BNC); (C) Mean inhibition zones (mm2) of different formulations of both, nanoparticles and BNC disks impregnated with nanoparticles, including negative controls without gentamicin sulfate and the statistically significant difference between groups (** = p < 0.003, *** = p < 0.001).
A one-way ANOVA revealed that there was a statistically significant difference in mean inhibition zones among the groups (p < 0.001). Subsequently, a post hoc Tukey’s HSD test was performed using the IBM SSPS Statistics Data Editor, revealing no statistically significant difference between the GS-Sol and BNC-GS-Sol (p = 0.970). Since the GS-Sol provides the antibiotic in a freely soluble form, it allows for rapid diffusion in the agar diffusion test, clearly producing the highest values for inhibition zones. These results also demonstrate that the BNC matrix does not hinder the release of the antibiotic. For both BNC-GS-Sol and GS-Sol, significantly larger inhibition zones were measured in comparison with GNP and BNC-GNP (p < 0.001). This reduction is likely attributable to the slower release rate and restricted diffusion of the encapsulated drug within the agar matrix compared to the rapidly diffusing free antibiotic. While the higher antimicrobial activity of free gentamicin compared to the nanoparticle-containing composites (GNP, BNC GNP) is consistent with the expected faster release of the free drug, direct quantitative data on release kinetics from the formulations are not included in the current study. Characterizing drug release profiles under physiologically relevant conditions is planned for future work and will better clarify the relationship between release behavior and antimicrobial efficacy.

3.5. In Vitro Studies

3.5.1. Cell Viability Studies

Cell viability was assessed via the MTT test and expressed as a percentage relative to the control group. A comparison between the three different formulations and the examined concentrations is presented in Figure 6. All formulations demonstrated viability exceeding the 70% threshold set by ISO-10993-5 [55]. indicating good biocompatibility and potential safety for biomedical applications. Notably, samples containing only Gentamicin sulfate showed excellent results. The observed slight decrease in viability with increasing concentration is anticipated and does not pose a significant cytotoxicity risk.
Figure 6. MTT assay results for BNC disks impregnated with gentamicin sulfate solution (BNC-GS-Sol), empty (BNC-NP), and gentamicin-loaded nanoparticles (BNC-GNP), containing 5, 10 and 15 μg gentamicin/disk. The figure includes pure BNC as a control.

3.5.2. Cell Adhesion

The micrographs (Figure 7) reveal distinct patterns of fibroblast BJ cells attachment and morphology on nanocellulose substrates. At lower magnifications (Figure 7A), the overall distribution of cells across the surface is visible, while higher magnifications (Figure 7B,C) reveal the cell–material interface, cell–cell bridging and morphological details at the cell–BNC disk interface.
Figure 7. SEM micrographs of fibroblast BJ cells attachment and morphology on the bacterial nanocellulose (BNC), nanocellulose impregnated with varying concentrations of gentamicin sulfate solution (BNC-GS-Sol), nanocellulose impregnated with empty nanoparticles (BNC-NP) and with gentamicin-loaded nanoparticles (BNC-GNP) at three different concentrations (ranging from 5 to 15 μg/disk) and magnification of (A) 1000× (scale bar = 20 μm), (B) 5000× (scale bar = 2 μm) and (C) 25,000× (scale bar = 1 μm).
A clear trend is visible across the concentration series. As the concentration increases from 5 to 15 μg/disk in all sample types (BNC-GS-Sol, BNC-NP, BNC-GNP), there is a noticeable reduction in the number of attached cells. The BNC-5G, BNC-5NP and BNC-5GNP samples show abundant, well-distributed cells with spindle-shaped morphology. The BNC-10G, BNC-10NP and BNC-10GNP samples display fewer cells with a more rounded appearance and the BNC-15G, BNC-15NP and BNC-15GNP samples exhibit the fewest attached cells, most with spherical morphology.
Despite these concentration-dependent effects, it is important to note that cell–nanocellulose interactions persist. The rounded cells visible in the highest concentration samples suggest active division processes occurring directly on the nanocellulose films. This indicates that while the quantity and quality of cell attachment may decrease with increasing concentration, the nanocellulose remains fundamentally biocompatible, supporting essential cellular functions including proliferation. Overall, these observations confirm that nanocellulose films support fibroblast growth across all tested conditions, though maintaining the fibrillar structure appears crucial for optimizing cell–material interactions [56].

4. Conclusions

This study presents the development and characterization of a bioactive BNC disk produced by BNC impregnation with gentamicin-loaded chitosan nanoparticles with suitable characteristics as potential wound dressings capable of simultaneously tackling the challenge of surgical-site infections (SSIs) and enabling improved wound healing. By optimizing the process and formulation parameters of the nanoparticles, a favorable particle size, distribution and zeta potential values were achieved, ensuring stability. The optimized nanoparticles demonstrated robust antibacterial activity against Klebsiella pneumoniae, highlighting their potential for local infection control. However, the authors acknowledge that surgical-site infections are often polymicrobial, and further studies evaluating the efficacy of the developed dressing against additional SSI-associated microorganisms are warranted. The nanoparticles’ impregnation onto the BNC disks did not compromise the structural integrity, maintaining the BNC desirable properties for cell attachment and proliferation, as observed by SEM analysis, while the in vitro studies performed on human fibroblast cell cultures revealed no significant cytotoxicity, proving their overall biocompatibility. Although these results are sufficient to underline the potential of the composite as a suitable wound dressing, further research is needed to fully explore the advantages and possible limitations. Namely, the biocompatibility, safety and efficacy should be validated in vivo, while the antimicrobial testing should be expanded against other clinically relevant bacterial strains. The optimization process could be enhanced by employing more complex experimental design models to fully capture the interactions between formulation and process variables, thereby ensuring improved performance and stability of the nanoparticles. Overall, these findings provide preliminary data supporting the advantages of utilizing multifunctional composite biomaterials for sustained, localized treatment of surgical-site infections, simultaneously ensuring enhanced tissue recovery.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/polym18040510/s1, Figure S1: Particle size distribution of the formulations where homogenization techniques were varied (NP-B-1 to NP-B-4) and formulations where the chitosan-to-TPP mass ratio was varied (NP-A to NP-F); Figure S2: Savitzky-Golay first-derivative analysis of the FTIR spectra in the 3050-2800 cm-1 (top) and 1800-1300 cm-1 (bottom) region of gentamicin-loaded nanoparticles (GNPs), empty nanoparticles (NPs), bacterial nanocellulose impregnated with NPs (BNC-15 NP), bacterial nanocellulose impregnated with GNPs (BNC-15 GNP) and blank BNC.

Author Contributions

Conceptualization, N.G., B.D. and S.G.; methodology, N.G., M.S.C., B.D., V.V. and S.G.; formal analysis, L.L.T., M.P., N.G., M.S.C., B.D., V.V., U.J. and S.G.; investigation, L.L.T., M.P., N.G., M.S.C., B.D., V.V., U.J. and S.G.; resources, N.G., M.S.C., B.D., V.V. and S.G.; writing—original draft preparation, L.L.T., M.P., V.V. and U.J.; writing—review and editing, N.G., M.S.C. and S.G.; visualization, L.L.T., M.P., B.D., V.V. and U.J.; supervision, N.G., M.S.C., B.D. and S.G.; project administration, B.D. and S.G.; funding acquisition, B.D. and S.G. All authors have read and agreed to the published version of the manuscript.

Funding

The authors would like to acknowledge the financial support from the Slovenian Research and Innovation Agency (ARIS) in frame of research core program group for Textile Chemistry P2-0118, project BI-ME/21-22-002 and N2-0388, and Ministry of education, science, culture and sports, Montenegro.

Data Availability Statement

Data is contained within the article or Supplementary Material.

Acknowledgments

Graphical abstract was created in BioRender. Gorgieva, S. (2026) https://BioRender.com/nyd8n9n, accessed on 15 February 2026.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

AbbreviationDefinition
BNCBacterial nanocellulose
BNC-GNPBacterial nanocellulose impregnated with gentamicin-loaded nanoparticles
BNC-GS-SolBacterial nanocellulose impregnated with gentamicin sulfate solution
BNC-NPBacterial nanocellulose impregnated with empty nanoparticles
CSChitosan
DMEMDulbecco’s Modified Eagle’s Medium
DMSODimethyl sulfoxide
DPBSDulbecco’s phosphate-buffered saline
FBSFetal bovine serum
GNPGentamicin-loaded nanoparticles
GSGentamicin sulfate
NPEmpty chitosan nanoparticles
TPPSodium tripolyphosphate

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