Novel chitosan-encapsulated ZnO NPs@Subtilisin: enzyme immobilized as next-generation nanoplatform for antimicrobial and anticancer control

Antibiotic resistance has become a major global health concern, significantly reducing the effectiveness of existing antimicrobial treatments. This issue, largely driven by the excessive and inappropriate use of antibiotics, has led to increased morbidity, longer hospital stays, and higher mortality rates. At the same time, lung cancer continues to be one of the deadliest cancers worldwide, with high incidence and poor survival outcomes. These challenges highlight the urgent need for innovative therapeutic approaches that can address both microbial infections and cancer. In this study, we developed a multifunctional nanosystem with both antibacterial and anticancer potential. Zinc oxide nanoparticles (ZnO NPs) were synthesized using eggshell membrane as a biological template. A targeted delivery system was then constructed by immobilizing the enzyme subtilisin onto the nanoparticle surface and encapsulating the complex within a chitosan matrix to improve stability and activity. The biological performance of the system was evaluated against A549 lung cancer cells and various bacterial strains, including Gram-positive and Gram-negative species. This study demonstrates that the ZnO/subtilisin nanocomposite had stronger antibacterial activity against E. coli and Pseudomonas aeruginosa than against Staphylococcus aureus and S. mutans . The results demonstrated strong inhibitory effects, attributed to mechanisms such as ion release, membrane interaction, and oxidative stress. This nanosystem shows promise as a dual-function therapeutic platform.

Zinc oxide nanoparticles (ZnO NPs) have attracted significant attention for biomedical applications due to their unique properties and multiple mechanisms of action. These particles have proven highly effective as antibacterial agents against both bacteria and cancer cells, making them a major focus of modern research (Lebaka et al., 2025; Hadi et al., 2025). Compared with other metal oxide NPs, ZnO NPs also demonstrate strong biomedical applications, including anticancer effects, drug delivery, antibacterial activity, diabetes therapy, anti-inflammatory activity, and wound healing. Due to ZnO’s excellent UV absorption properties, it is widely used in personal care products, including cosmetics and sunscreen (Łukowiak and Stolarczyk, 2026; Kapare et al., 2025). Lung cancer is the second most common form of cancer and the leading cause of cancer death. Most cancer deaths are attributed to the metastatic spread of cancer to distant organs. Pulmonary neoplasms metastasize more frequently to bone, brain, lung, and liver, resulting in shorter survival. For patients with non-metastatic lung cancer, median survival after diagnosis was 13 months, and for metastatic disease, 5 months. The metastatic sites and survival in patients with metastatic lung cancer vary according to sex, histological subtype, and age at diagnosis (Zhang et al., 2025; Wang et al., 2025). These arise from the neoplastic transformation of type I1 alveolar cells. The A549 cell line was derived in 1972 by Giard et al. from the resection and culture of malignant lung tissue from a 58-year- old Caucasian man with a pathologically proven tumor. They are human alveolar basal epithelial cells with a squamous morphology that perform the diffusion function of returning water and electrolytes to the lungs from the alveoli. A549 cells, Human alveolar basal epithelial type II cells, have an adherent growth pattern and form monolayers. A549 cells were derived from the neoplastic transformation of type I1 alveolar cells (Shahzadi et al., 2023). Subtilisin, a highly efficient BAP (broadly active proteolytic enzyme), is a widespread protease of the serine class found all over and secreted by different species of Bacillus, owing to its ability to hydrolyze peptide bonds under diverse physiological conditions. While traditionally used in industrial applications, the potential biological properties of this enzyme have attracted interest in the scientific community, as it may be useful in medical applications. Studies suggest subtilisin exhibits some antibacterial activity in biofilm disruption and a potent ability to inhibit the growth of certain tumoral cells (Shettar and Bagewadi, 2025; Haldar et al., 2025; Shettar et al., 2025). These characteristics reveal novel opportunities for using subtilisin as a potential treatment or clinical aid in current medical settings. It is used to formulate therapeutic ointments for cleansing wounds and ulcers by separating damaged tissue from healthy tissue with minimal injury to healthy cells. It is an alkaline protease that can withstand high pH and temperature levels (Mathioudaki et al., 2024). It has become a crucial component in bio-detergents formulations and advanced biotechnological processes (Mechri et al., 2022). Because of growing bacterial resistance to antibiotics and difficulties in controlling infections with current antibiotic classes, there is an urgent need for effective antibacterial molecules and alternatives to chemical antibiotics. Also, the properties of proteins and peptides, such as anticancer, antioxidant, anti-inflammatory, antimicrobial, and antiparasitic activity, make them promising biomolecules for healthcare applications (Ahmed et al., 2024). The green synthesis of nanomaterials is a novel, low-cost, and eco-friendly process in which naturally derived materials such as plants, bacteria, fungi, and enzymes replace toxic chemical reagents. It reduces toxic waste generation, operates under mild conditions, and typically uses biological reducing agents; thus, it is considered a sustainable method for synthesizing nanomaterials with biomedical and industrial applications (Saxena et al., 2025; Protik et al., 2025). Chitosan (CS) is a biocompatible, non-toxic amino polysaccharide with unique properties, including mucoadhesive activity, permeation-enhancing effects, and antitumor and antimicrobial activity. Chitosan’s antimicrobial activity depends on interactions between its positively charged amino groups and the negatively charged surfaces of bacterial cells (Harugade et al., 2023; Guarnieri et al., 2022). This interaction may cause leakage of cellular components and eventually cellular damage. These implementations of CS and subtilisin on ZnO NPs led to the development of an advanced antibacterial nanocomposite with complementary effects (Raafat et al., 2008). ZnO NPs generate reactive oxygen species (ROS) and disrupt the cell wall by damaging the phospholipid bilayer and inducing intracellular leakage. CS enhances the stability and antibacterial activity of ZnO NPs in the nanocomposite. At the same time, subtilisin reduces biofilm thickness and disassembles its structure, increasing treatment-system permeability and improving efficiency against bacterial cells (Chopra et al., 2025). ZnO nanoparticles have a remarkable biomedical potential. Their translation into practical biomedical applications is frequently constrained by inherent physicochemical instability; they tend to aggregate under physiological conditions. Various surface passivation strategies have been developed to avoid these limitations, with increasing emphasis on approaches that circumvent harsh chemical treatments. PEGylation, for instance, has been widely adopted to improve both the colloidal stability and biological compatibility of ZnO NPs. However, it lacks the strong covalent anchoring required for prolonged stability in harsh conditions. In a different vein, polydopamine coatings have garnered considerable interest as versatile bio-inspired crosslinkers, they suffer from oxidative instability. More recently, the incorporation of ZnO NPs into polymeric networks such as double-network hydrogels—comprising, for example, polyacrylamide and carboxymethyl chitosan—has been investigated to achieve not only mechanical reinforcement but also controlled liberation of therapeutic zinc ions. However, this approach necessitates complicated fabrication and sequential crosslinking. Each strategy offers distinct advantages, yet it often involves multistep procedures or requires sophisticated conjugation chemistries. Our study employs chitosan, a naturally abundant, biodegradable, biocompatible polymer, which functions as both a stabilizing matrix and a versatile anchoring layer. Furthermore, glutaraldehyde, used as a mild crosslinking agent, facilitates controlled enzyme immobilization without requiring hazardous silane coupling agents. Our study is a simple, cost-effective, and efficient approach to nanoparticle stabilization. In this way, the nanocomposite is intended to harness the stabilizing function of chitosan alongside the biofilm-degrading and potentially antitumor properties of subtilisin. This provides a comparatively straightforward route to a multi-action nanoplatform (Poojar et al., 2026; Guo et al., 2026). The combination of CS and subtilisin on ZnO NPs yields a versatile nanocomposite with both antibacterial and anticancer efficacy: subtilisin delivers a boosted therapeutic assault, and CS confers stability and prolonged antibacterial activity. At the same time, ZnO NPs generate reactive oxygen species (ROS) and disrupt bacterial membranes (Akdaşçi et al., 2025). We hypothesize that a chitosan-encapsulated ZnO/subtilisin nanosystem exhibits enhanced dual antibacterial and anticancer activity compared to ZnO nanoparticles alone, through synergistic effects involving enzyme immobilization, improved stability, and increased cellular interaction. Therefore, developing multifunctional nanocomposites with stability, antibacterial, and anticancer efficacy represents a potential strategy. In this study, a hybrid nanocomposite consisting of a ZnO NP core encased in a chitosan layer was prepared, to which the enzyme subtilisin was linked as a proteolytic agent. The manufactured nanocomposite was then characterized using transmission electron microscopy (TEM), ultraviolet-visible spectroscopy (UV-Vis), and Fourier transform infrared spectroscopy (FTIR). The antibacterial efficacy of the manufactured ZnO/subtilisin nanocomposite against Gram-negative and Gram-positive bacteria was evaluated. Different assays were performed to investigate the anticancer activity of the ZnO/subtilisin nanocomposite against lung cancer cell lines (A549).

Synthesis of zinc oxide nanoparticles

Eggs were purchased from the local market. They were gently cracked, and the membranes were manually separated from the shells. The membranes were first washed with water, then immersed in a 20% acetic acid solution for 3 h to ensure the complete removal of the calcium carbonate (CaCO 3) shell. Afterward, the membranes were rinsed again with distilled water. A precursor solution was then prepared by dissolving 3.7 g of zinc nitrate (Zn(NO 3) 2 ·6H 2 O) in 70 mL of deionized water, with continuous stirring on a magnetic stirrer for 30 min. The egg membranes were immersed in this solution, allowing Zn +2 cations to deposit onto their surfaces. After 24 h, the membranes were removed and dried at room temperature. They were then heat-treated in an oven at 700 °C for 3 h, yielding a fine white powder (Camaratta et al., 2015).

Preparation of the ZnO NPs/chitosan/glutaraldehyde/subtilisin

The preparation process followed (Soares et al., 2020), with minor modifications. Chitosan (62.5 mg) was dissolved in 12.5 mL of deionized water, then 1% (v/v) acetic acid was added under stirring to aid dissolution. 155 mg of ZnO NPs was then added to the chitosan solution and stirred for 30 min. The resulting mixture was ultrasonicated for 10 min to ensure uniform dispersion. Subsequently, 10 mL of NaOH solution (5 mg/mL) was added dropwise to the mixture. This step precipitated chitosan onto the surface of the ZnO particles. The precipitate was collected, washed several times with ultrapure water, and centrifuged at 4,000 rpm for 10 min per cycle (three wash cycles) until the pH of the supernatant reached approximately 6.5–7.0, then dried at 50 °C for 24 h. The dried product was then dispersed in 5 mL of a 2.5% (v/v) glutaraldehyde solution and stirred continuously at room temperature for 1.5 h to activate the surface. After activation, the composite was thoroughly washed with deionized water to remove any unreacted glutaraldehyde. In the final step, the activated carrier was dispersed in 8.25 mL of deionized water, followed by the addition of subtilisin enzyme solution (3 mg/mL). The mixture was kept under continuous stirring at room temperature for 2 h to allow enzyme immobilization onto the composite surface. To investigate the morphology of the produced nanoparticles and nanocomposites, a transmission electron microscope (TEM) was used; the produced nanoparticles’ XRD patterns were obtained using an XRD (Panalytical X' Pert Pr United Kingdom). The measurements were performed with Cu Kα radiation (λ = 1.54060 Å) generated at 40 kV and 30 mA. The diffraction data were collected over a 2θ range of 10.02°–79.98° using continuous-scan mode with a step size of 0.0400° and a scan step time of 0.25 s, corresponding to a scan rate of 0.16°/s (9.6°/min). The divergence slit and receiving slit were set at 1.00° and 0.10 mm, respectively, and all measurements were carried out at room temperature (25 °C). The optical characteristics of the resulting compound were measured with a UV–Vis spectrophotometer (Shimadzu UV-1800, Kyoto, Japan). Fourier transform infrared (FTIR) spectra and Energy-dispersive X-ray spectrometry (EDS) mapping tests were performed to determine the functional groups and the elemental composition of ZnO NPs and the ZnO/subtilisin nanocomposite.

Anticancer activity of ZnO NPs and ZnO NPs/Subtilisin nanocomposite

MTT assay

This assay was performed to evaluate the toxic effect of the ZnO NPs and ZnO NPs/subtilisin nanocomposite. A549 and PCS-201–013 cells were purchased from Sigma (United States of America). A549 cells (1 × 10 4 cells/well) were incubated for 24 h at 37 °C after culturing in a 96-well plate with RPMI-1640 medium. Then, cells were treated with ZnO NPs and ZnO NPs/subtilisin at different concentrations (12.5, 25, 50, and 100 μg/mL) for 48 h; MTT solution (Sigma–Aldrich, United States of America) at 3 mg/mL was added to each well. After aspirating the medium, DMSO (100 μL) (Sigma–Aldrich, United States of America) was added. A microplate reader was used to measure the absorbance of all samples at 492 nm (Kadhim et al., 2026).

Changes in nuclear morphology

DAPI (Sigma–Aldrich) fluorescent stain was used to evaluate nuclear changes in A549 cells exposed to ZnO NPs and a ZnO NPs/subtilisin nanocomposite. Cells were plated in a 96-well plate at 1 × 10 4 cells per well and incubated with 10 μg mL -1 of each prepared compound. Afterward, they were washed twice with PBS and fixed with absolute ethanol. Following a 30-min incubation with ethanol, the cells were washed again in PBS. DAPI staining solution was added to each well, and the cells were incubated for 20 min. A fluorescence microscope was then used to identify alterations in nuclear morphology.

Acridine orange–propidium iodide staining

A549 cells were seeded into a 96-well plate at 1 × 10 4 cells per well. After 1 day of incubation, 25 μg mL -1 of ZnO NPs and a ZnO NPs/subtilisin nanocomposite were transferred to all wells. After 24 h of incubation, the cells were washed twice with PBS. Cells were stained using 200 μL of AO/PI at 5 μg/mL. We used a fluorescence microscope to examine nuclear changes in the stained cells.

Genotoxicity assay

A comet assay was conducted to investigate DNA damage in cells exposed to ZnO NPs and a ZnO NPs/subtilisin nanocomposite. After A549 cells were exposed to 10 μg mL −1 of the prepared compounds, they were resuspended in low-melting-point agarose (LMAgarose, Trevigen, United States of America) at 1 × 10 5 cells/mL at 37 °C in a 1:10 (v/v) ratio. Next, the sample was applied to a comet slide and incubated in lysis solution for 1 h at 4 °C. The slides were rinsed with distilled water and placed in alkaline unwinding solution at RT for 20 min. Subsequently, the slides were transferred to an electrophoresis tray that contained NaOH-EDTA solution (Sharma et al., 2025).

Antibacterial activity of ZnO NPs and ZnO NPs/subtilisin nanocomposite

The antibacterial effect of ZnO NPs and ZnO NPs/subtilisin nanocomposite against Gram-positive Staphylococcus aureus, S. mutans, and Gram-negative E. coli and Pseudomonas auroginosa bacteria was investigated by the well diffusion assay. Plates containing Mueller-Hinton agar were prepared and inoculated with bacteria. Wells were created in the agar medium with a diameter of around 6 mm. ZnO NPs and a ZnO NPs/subtilisin nanocomposite were transferred to the wells at concentrations of 33.1, 62.5, 125, and 250 μg/mL, while wells of the negative control were loaded with distilled water. All cultures were plated and incubated overnight at 37 °C. The diameter of the inhibition zone was determined using a ruler (Kadhum et al., 2024).

Determination of MICs and MBCs of ZnO NPs and a ZnO NPs/subtilisin

MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) assays were conducted to evaluate the antibacterial effect of ZnO NPs and a ZnO NPs/subtilisin nanocomposite against Gram-positive S. aureus, S. mutans, and Gram-negative E. coli and P. auroginosa bacteria, which were inoculated in nutrient broth and incubated for a day at 37 °C. The obtained cultures were adjusted to a 0.5 McFarland standard turbidity. Each well of a 96-well sterile microtiter plate was loaded with 100 µL of aseptic nutrient broth. Then, 100 µL of ZnO NPs and a ZnO NPs/subtilisin nanocomposite were transferred to each well. To confirm assay reliability, all treatments were performed in triplicate. Suspensions of standardized bacteria (10 μL) were transferred to the wells, while the negative control well contained only aseptic broth. After incubation for a day at 37 °C, each well was loaded with 20 µL of resazurin solution to assess bacterial viability. The plates were incubated in the same environment for another 4 hours. MIC is the lowest concentration of the prepared compound required to inhibit visible bacterial growth. Subculturing samples from a clear MIC well on agar confirmed the concentration that prevents bacterial growth, defined as the lowest concentration of the prepared compound that did not form colonies (Dayekh et al., 2025).

ROS production assay

Intracellular ROS can oxidize nonfluorescent, cell-permeable DCF-DA to a fluorescent product. The DCF Fluorescence signal is linearly related to ROS generation. The bacteria were then exposed to 125 μg/mL ZnO NPs and 125 μg/mL ZnO NPs/subtilisin nanocomposite. They were then stained with 10 μM DCF-DA for 1 h 15 min. The bacteria were then rinsed with 0.85% saline solution. Flow cytometry was then used to analyze DCF fluorescence intensity across all samples.

Statistical analysis

In this work, GraphPad Prism (V7) was utilized to perform Statistical analyses of the Data. Group comparisons were performed using one-way ANOVA. Hence, Tukey’s post hoc test was performed to determine which groups differed significantly. A p-value less than 0.05 is regarded as statistically significant. Data are shown as mean ± standard deviation of three replicate experiments.

Characterization of ZnO NPs and ZnO NPs/subtilisin nanocomposite

Transmission electron microscopy (TEM) was also used to observe the surface state of ZnO nanoparticles and further determine their morphology. The samples are well-defined as three-dimensional nanoparticles in the upper panel of Figure 1A (upper panel). TEM images of the ZnO powder sample prepared via the green synthesis route show uniform nanorods. Figure 1B (upper panel) shows the ZnO NPs/subtilisin nanocomposite after immobilizing subtilisin on ZnO NPs. A comparison of the images A and B in Figure 1 (upper panel) demonstrates a difference in size between the ZnO NPs and the ZnO NPs/subtilisin nanocomposite, where the observed increase in thickness is attributed to the coating of ZnO NPs by subtilisin. Studies have confirmed that zinc nitrate is the most effective material for accurately mimicking biomolecules, resulting in uniformly shaped nanoparticles (Dong et al., 2007; Pandas and Fazli, 2018). X-ray diffraction (XRD) is an important technique for studying structural properties and identifying nanomaterials. The results of XRD of ZnO NPs@Eggshell prepared by the chemical precipitation method showed the presence of XRD angles at the crystal planes of (100) (002), (101) (102), (110) (103), (200) (112), and (201), as shown in the lower panel of Figure 1 (lower panel). Sharp, narrow diffraction angles were observed with no other impurities, confirming the high crystallinity of the NP structures and enhancing the purity of the prepared sample. While the crystal system was hexagonal, these results are consistent with the standard card. Reference code: 01–079-2205.

To calculate the crystal size (CS), we apply the Scherrer equation (Equation 1). θ is the Bragg angle, λ is the X-ray wavelength in nm, β is the line broadening at half the maximum intensity (FWHM), and K is a dimensionless shape factor equal to 0.9. The results show that the average CS was 39 nm, as shown in Table 1. This result reflects the small size of ZnO crystals. The XRD pattern of ZnO NPs/Subtilisin displayed diffraction peaks (100), (002), (101), (102), (110), (103), (200), (112), (201), and (202) planes of hexagonal wurtzite ZnO, as illustrated in the lower section of Figure 1(lower panel). No impurity phases were identified. The retention of distinct ZnO diffraction peaks after surface functionalization indicates that the crystalline structure of ZnO remained unchanged throughout the coating and enzyme immobilization processes.

FTIR spectra were recorded to identify functional groups and possible interactions. Figure 2A shows the FTIR spectra of ZnO NPs; the main band appeared at 402 cm -1 corresponding to the Zn-O stretching vibration, and another band was detected at 1,131 cm -1, corresponding to the bending vibration of C-O. At the same time, the FTIR of the ZnO NPs/subtilisin nanocomposite indicates the presence of bands at 3,374 cm -1, 1,376 cm -1, and 1,032 cm -1, corresponding to O-H and N-H stretching, C-H bending, and C-O stretching vibrations, respectively (Parlakyigit et al., 2023; Tabe et al., 2026). These results corroborate the chemical binding mechanism, in which glutaraldehyde should bind to chitosan amino groups on one side and to enzyme amino groups on the other, forming a stable coating layer. This process creates Schiff bases, which are involved in enzyme stabilization (Rajesh et al., 2018). Taken together, these FTIR results support successful surface modification of the ZnO nanoparticles (Figure 2B).

In a further confirmatory test, the absorption peak of the nanoparticles appears at 374 nm, confirming the presence of ZnO NPs, as illustrated in Figure 3A. Figure 3B shows a notable blue shift of the absorption peak to approximately 280 nm and a reduction in intensity after immobilizing subtilisin on ZnO NPs. The observed blue shift is ascribed to interactions between the organic coating layer and ZnO nanoparticles, with band-gap widening resulting from perturbations in the electronic structure. The reduction in the absorption peak intensity due to the organic shell on ZnO NPs restricts light penetration and enhances scattering. All these spectral changes together establish the successful coating of ZnO nanoparticles with subtilisin (Lakshmi et al., 2024; Bel Haj Mohamed et al., 2014).

Energy-dispersive X-ray spectrometry (EDS) mapping was performed to determine the elemental composition. Figure 4 illustrates the uniform distribution of elements Zn, O, C, and Cl throughout the ZnO NPs/subtilisin nanocomposite. These data show a notable difference in elemental composition after coating ZnO NPs with the enzyme, confirming the successful immobilization of subtilisin on ZnO NPs. Carbon is the primary indicator of the enzyme’s presence, and its distribution alongside zinc indicates that the enzyme surrounds the particles. The presence of chlorine is another indicator that the enzyme retains its natural structure, as these ions bind to it to maintain its electrical charge. This proves that the enzyme was not damaged during the encapsulation process. The presence of chlorine (Cl) in the EDX in Figure 4B is not contamination. Structural biology studies found that chloride ions bind specifically to the subtilisin enzyme through its natural interactions with the environment. It would be expected that charged enzyme groups are more closely associated with counterions when bathed in low-dielectric, nonaqueous media (Gorohovs et al., 2025; Cianci et al., 2010). The EDS spectrum revealed a distinct peak of Zn, indicating the central ZnO composition, while the O peak confirms its oxidized state, as indicated in Figure 5. The strong Zn signal appeared at 1 keV, while C was indicated at approximately 0.27 keV. These data align with previous studies (Shettar et al., 2024). Figure 6 represents the zeta potential results of ZnO NPs and ZnO NPs/Subtilisin. ZnO NPs showed a negative surface charge with a zeta potential of almost −14 mV, indicating good colloid stability (Upper panel). ZnO NPs/Subtilisin showed a positive surface charge with a zeta potential of almost 18 mV, indicating successful enzyme immobilization and good colloid stability (Lower panel). Real binding of the Subtilisin to the surface of ZnO NPs was exhibited via quantitative exploration of Subtilisin immobilization. We used the BCA method to measure protein concentration according to the manufacturer’s protocol. The concentration of Subtilisin before immobilization was 0.5 mg/mL. After immobilization, the concentration decreased to 0.093 mg/mL, as indicated in Table 2, indicating an immobilization efficiency of 81.4%.

Anticancer activity

Cytotoxicity of ZnO NPs and ZnO NPs/Subtilisin against lung cancer cells

To study the kinetics of the ZnO/subtilisin nanocomposite, UV-Vis spectroscopy was used at pH 5.3 and 7.4. The release of Subtilisin was almost 75% at pH 7.4 and almost 90% at pH 5.3 after 24 h, as indicated in Figure 7. The anticancer activity of ZnO NPs and the ZnO/subtilisin nanocomposite against lung cancer cells, and their ability to suppress cancerous cell growth, were evaluated after treatment. The results show that cancer cell viability was considerably reduced when the culture medium was supplemented with the prepared compounds (P ≤ 0.01 and P ≤ 0.001, respectively). As shown in Figure 8 (upper panel), the toxicity of ZnO NPs and the ZnO/subtilisin nanocomposite increased with increasing concentration, reaching approximately 77.16% for ZnO NPs and 85.5% for the ZnO/subtilisin nanocomposite at the same concentration of both compounds. These percentages indicate that the ZnO/subtilisin nanocomposite was more toxic to A549 cells than ZnO NPs alone. ZnO/subtilisin nanocomposite exhibits higher cytotoxicity when compared with pure ZnO NPs against A549 cells. This is due to the synergistic effect of the subtilisin and nanoparticles. In contrast, ZnO NPs alone and the ZnO/subtilisin nanocomposite show low cytotoxicity against normal human cancer cell lines, as shown in Figure 8 (lower panel). The nanoparticles produce more free radicals, and binding to the enzyme facilitates their crossing of the cell membrane. Therefore, the coupling of these two results in large amounts of ROS, which are considerably higher than those induced by the nanoparticles alone, thereby accelerating cancer cell death by apoptosis. Additionally, the compound may potentiate multiple pathways for inducing cancer cell death (Jankowski et al., 2025; Zhao et al., 2023). However, other studies also demonstrate that nanoparticles can cross the cell membrane owing to their surface properties. This is often seen in functionalization or the attachment of enzymes/molecules to a surface; for example, when particles are coated with chitosan, which imparts a positive charge that facilitates binding to the negatively charged cell membrane (Payomhom et al., 2024; El-Saadony et al., 2025). While enzymes alone have not been shown to diffuse across the cell membrane, nanoparticles coated with or conjugated to proteins or enzymes can enter cells via endocytosis, a cellular uptake mechanism, and other pathways are regarded as the primary means of delivering nanoparticles into cells (Wang et al., 2024).

Assessment of nuclear morphology

To study the effects of the ZnO NPs and ZnO/subtilisin nanocomposite on nuclear morphology and measure the viability of cancer cells, fluorescent staining (DAPI) and AO/PI were used. AO/PI were used to study cell viability and nuclear alterations in apoptosis (Chauhan et al., 2025). Cells were exposed to ZnO NPs and ZnO/subtilisin nanocomposite at 25 µgmL -1 per well. The results showed that dead cells fluoresced orange or red, while live cells fluoresced green. As demonstrated in Figure 9, there was a rise in plasma membrane damage and vacuoles during lysosome formation in the cells exposed to the compounds in comparison with control cells. PI is a stain that penetrates only damaged or dead cells, whereas AO is a stain that penetrates all cells (both living and dead). The AO/PI stain not only differentiates between living and dead cells but can also provide a preliminary indication of the cell death pathway, as distinctive nuclear signs such as chromatin condensation or nuclear fragmentation can be seen, which are attributes of apoptosis. A yellow color denotes the early stages of apoptosis, due to changes in permeability and the interaction of the two stains (Wang et al., 2023; Payomhom et al., 2025). Nuclear morphology was examined after treating cancer cells with the synthesized compounds and staining with DAPI. As shown in Figure 10, DAPI staining indicated that treatment of 549 cells with ZnO NPs resulted in morphological features characteristic of apoptosis, such as nuclear fragmentation, micronuclei formation, and chromatin condensation, compared with control cells, which remained round. By contrast, the treated cells displayed the morphological features of early apoptosis. These features included brightly blue-staining apoptotic bodies and pyknotic nuclei. Cell morphology began to change after 24 h of treatment. Cells treated with the ZnO/subtilisin nanocomposite were more fragmented, and apoptotic cells were observed. Some cells exhibited nuclear fragmentation, a hallmark of apoptosis. The rounded cells had aggregated, and most were in late apoptosis (Ibrahim et al., 2018; Mir et al., 2025).

A comet assay was performed to investigate DNA damage in lung cancer cells induced by ZnO NPs and the ZnO/subtilisin nanocomposite, using tail DNA parameters. The comet assay is a common experimental method for evaluating DNA damage, in which cells with DNA damage exhibit increased migration of nucleic acids toward the anode. This assay was selected for its sensitivity to detect minimal DNA damage, which can be caused by low concentrations of nanoparticles (Tyutereva et al., 2024; Bajpayee and Dhawan, 2026). In this work, the results show no comet formation in untreated cells, as illustrated in the upper panel of Figure 11A. When A549 cells were exposed to the prepared compounds, nucleoid bodies migrated away to form comets, indicating DNA damage (He et al., 2025). It is noteworthy that the tail length increased considerably in the A549 cell line that was treated with ZnO NPs and ZnO/subtilisin nanocomposite, as illustrated in Figure 11. ZnO NPs and the ZnO/subtilisin nanocomposite triggered DNA fragmentation, which can lead to apoptosis and cell death. The results also showed that longer DNA tails appeared when cells were treated with the ZnO/subtilisin nanocomposite (Vandghanooni and Eskandani, 2011; Abrica-González et al., 2025). In untreated cells, nucleic acids do not fragment. Nanomaterial exposure can alter cellular function, disrupt the genome, and cause organ-specific toxicities. Therefore, alternative toxicity mechanisms need to be explored to inform safety considerations for such nanoparticles and human health (Abegunde et al., 2025; Encinas-Gimenez et al., 2024). Table 3 summarizes nanocomposites and their anticancer activity against different cell lines.

Antibacterial activity

Antibacterial activity of ZnO NPs and the ZnO NPs/subtilisin

In this study, we evaluated the antibacterial activity of ZnO NPs and the ZnO NPs/subtilisin nanocomposite against E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, and S. mutans. The zones of inhibition were measured and reported; the ZnO NPs/subtilisin nanocomposite exhibited the largest inhibition zone against all tested bacterial isolates. The zones measured 21.67 ± 0.9939 mm for P. aeruginosa, 20.68 ± 0.8083 mm for E. coli, 19.89 ± 0.8227 mm for S. mutans, and 19.49 ± 0.5792 mm for S. aureus, as shown in Figures 12–15. The results demonstrated that the zone of inhibition for the ZnO NPs/subtilisin nanocomposite is considerably higher compared with those obtained by ZnO NPs alone. At physiological pH, the ZnO NPs/subtilisin nanocomposite has a positive charge, which interacts with negatively charged molecules on the bacterial cell surface (Zubair et al., 2025; Huang et al., 2020). This facilitates penetration by disrupting the cell membrane, killing bacteria efficiently (Girma et al., 2024). ZnO NPs/subtilisin nanocomposite proved to have a higher antibacterial effect toward Gram-negative bacteria compared with its activity against Gram-positive bacteria due to structural differences in the outer membrane between Gram-positive and Gram-negative bacteria. Compared with Gram-positive bacteria, Gram-negative bacteria have a more complex cell envelope because of an additional outer membrane, a thinner peptidoglycan layer (7–8 nm), and an outer envelope containing LPS. Nanoparticles target a specific part of this membrane, increasing its permeability. Therefore, Gram-negative bacteria are susceptible to compounds that typically inhibit the outer membrane. However, this group of bacteria differs from Gram-positive bacteria, which have a thick peptidoglycan wall (20–80 nm) that confers resistance (Raina and Klein, 2025). Gram-negative bacteria have an outer membrane that these enzyme-coupled nanoparticles can target or more easily penetrate (Lee et al., 2024). Other studies have shown that chitosan has antibacterial properties. Because of its positive charge at physiological pH, CS interacts with negatively charged bacterial membranes, disrupting the cell membrane. Concurrently, subtilisin induces proteolytic degradation of cell membrane proteins, resulting in loss of control over cell permeability and leakage of internal contents, ultimately leading to cell death (Gastrell and Vollmer, 2025; Wu et al., 2024).

MIC and MBC values

The antibacterial effectiveness of both ZnO NPs and the ZnO NPs/subtilisin nanocomposite was also evaluated by MIC and MBC tests against the Gram-positive Staphylococcus aureus and S. mutans and Gram-negative E. coli and Pseudomonas auroginosa. The minimum inhibitory concentration (MIC) is the lowest concentration that prevents visible bacterial growth. MIC is used to compare the antimicrobial effects of various compounds by determining how increasing concentrations of an antibiotic or antiseptic affect microbial growth inhibition over a defined period. The MBC is defined as the lowest antimicrobial concentration that decreases the viability of the original bacterial inoculum by a specified amount (Wei et al., 2025; Kadeřábková et al., 2024). A moderate antibacterial activity of ZnO NPs was detected, as illustrated in Figure 16A,C,E,G and Table 4, with MIC and MBC values of 15.65 and 31.25 μg/mL. Significantly, the ZnO NPs/subtilisin nanocomposite displayed in Figure 16B,D,F,H greatly improved potency, with MIC values as low as 7.812 μg/mL for S. aureus and S. mutans. ZnO NPs/subtilisin nanocomposite has increased potency compared with bare nanoparticles due to synergistic effects from the CS polymer and enzymes that can increase adhesion, reactive oxygen species (ROS) production, ion release, and lessen agglomeration. Positively charged chitosan has NH 3 + groups that bind strongly to negatively charged bacterial cell walls, leading to Membrane disruption, increased membrane permeability, and allowing more Zn 2+ ions and subtilisin to enter the bacterial cell (Abu-Gharbia et al., 2025). These findings are consistent with earlier studies of CS–ZnO nanocomposites exhibiting effective antibacterial activity at low doses, due to the combined effects of membrane damage and enzyme delivery. Subtilisin is a serine protease that cleaves biofilm matrix proteins and bacterial surface proteins, creating pores (Yan et al., 2021). Notably, Gram-negative bacteria are less resistant because their complex cell membrane is composed of a thin peptidoglycan layer. This structure functions as a barrier restricting the entry of many materials. Taken together, the results verify that integrating ZnO NPs, CS, and subtilisin into a single nanocomposite reduces the antibacterial dose and improves treatment efficacy. The outcomes suggest that the prepared composite may be less toxic and more effective than traditional antibacterial agents (Liu et al., 2021). A previous study suggests that chitosan nanoparticles and zinc oxide nanoparticles (ZnO NPs) have been shown to have higher efficacy against Gram-negative bacteria, E. coli and E. cloacae, than Gram-positive ones, S. mutans and S. aureus, mostly due to the thinness of the peptidoglycan layer in Gram-negative bacteria, which facilitates nanoparticle penetration (Zubair et al., 2025). Bacterial biofilm formation on devices and hospital surfaces promotes bacterial spread and microbial and bacterial strain resistance (Asokan et al., 2026a; Asokan et al., 2026b). The ZnO NP-coated ETTs were tested against Klebsiella pneumoniae and showed antibacterial activity, reducing bacterial colonies by almost 47.6% and reducing biofilm formation by 41.5% (Asokan et al., 2025). Another study demonstrated that biogenic ZnO NPs mediated by Bacillus bacteria-controlled ESBL-producing K. pneumoniae (Asokan et al., 2026c). Meanwhile, ZnO NPs mediated by leaf extract of Thespesia populnea have multifunctional therapeutic effects, including antioxidant, cytotoxic, and anti-inflammatory activities (Kurien et al., 2025). A recently published study demonstrated the role of green-synthesized ZnO NPs in different medical applications (sokan et al., 2026).

Bacterial ROS generation

ROS generation in bacterial strains after being treated with ZnO NPs and ZnO NPs/Subtilisin is represented in Figure 17. ROS generation was measured using the DCFDA assay. Flow cytometry results indicated that the untreated bacterial control group exhibited minimal ROS production. In contrast, treatment with ZnO NPs and ZnO NPs/Subtilisin at 125 μg/mL resulted in a marked increase in ROS levels. The results showed that ROS production levels in Gram-negative bacteria E. coli and Pseudomonas aeruginosa were higher than in Gram-positive bacteria Staphylococcus aureus and S. mutans. A study showed that PEG-functionalized ZnO–CuO nanocomposites exhibited strong antibacterial activity, and that responses against Gram-negative bacteria E. coli, P. aeruginosa, and Klebsiella pneumoniae were higher than against Gram-positive S. aureus in many tests because the particles interact more easily with the outer membrane of Gram-negative bacteria (Jayanetti et al., 2024). Table 5 summarizes nanocomposites and their antibacterial activity against different bacterial strains.

In this study, ZnO NPs were created via an eco-friendly, inexpensive method using eggshell membrane. We designed and synthesized a novel nanoplatform by immobilizing the subtilisin enzyme onto ZnO NPs. These nanocomposites exhibited high efficacy in inhibiting A549 cell proliferation and bacterial growth, as confirmed in this study through multiple assays, compared with nanoparticles alone. The enhanced activity of the designed ZnO NPs/subtilisin nanocomposite is attributed to the unique properties of its components. Numerous studies have demonstrated the significant role of nanoparticles as antibacterial and anticancer agents, mediated through the generation of reactive oxygen species and disruption of the plasma membrane. This observation may be explained by the combined action of subtilisin, which degrades surface proteins, and the positively charged nanocomposite, which disrupts the bacterial membrane. Glutaraldehyde prevents enzyme leaching from the nanoparticles by forming stable covalent bonds. The findings suggest that the resulting nanocomposite holds promise as a therapeutic agent for lung cancer and as an antibacterial strategy to mitigate the risk of antibiotic resistance.