Multifunctional chitosan nanoparticles: Zn²⁺ adsorption, antimicrobial activity, and promotion of aquatic health

2.1 Synthesis of CSNPs

The CSNPs were synthesized using the ionotropic gelation method, with minor modifications to the concentrations of CS and sodium tripolyphosphate (STPP). Initially, 3 g of CS powder (commercially purchased) was set aside, while a solution of 1 mL acetic acid mixed with 800 mL of distilled water was prepared and stirred with a magnetic stirrer for 20 min. The CS powder was gradually added to the stirring solution, and the mixture was maintained at 450 rpm. To ensure optimal conditions, the pH was adjusted to 4–5 by adding a sodium hydroxide (NaOH) solution prepared by diluting two NaOH pellets in purified water. This solution was stirred continuously for 12 h to achieve complete dissolution and stability of the CS. Concurrently, an STPP solution was prepared by dissolving 0.40 g of STPP in 100 mL of distilled water using a motorized stirrer for 30 min to ensure complete dissolution. Subsequently, the STPP solution was added dropwise to the CS solution under instantaneous magnetic stirring for 40 min, creating a 0.4% (w/v) STPP solution. The resulting CS–TPP solution was centrifuged at 8,000 rpm and 4°C for 45 min, and the resulting pellets were washed thoroughly with distilled deionized water to remove impurities. The cleaned pellets were then dried in a hot air oven and stored for further use. These dried CSNPs were characterized for their properties and utilized in feed preparation, demonstrating their potential for practical applications.

2.2 CSNP characterization

The CSNPs were characterized using UV–visible spectroscopy (Shimadzu, Japan) in the range of 200–500 nm to find the maximum absorption peak to confirm preliminary formations. Fourier transform infrared (FTIR) spectroscopy (Avatar330, Thermo Nicolet) was employed to identify the surface functional groups in the range of 4,000–400 cm⁻¹ via the KBr pellet method. Powder X-ray diffraction (XRD; Siemens D5000 X-ray diffractometer) was utilized to obtain the diffraction peaks of the CSNPs in the 2θ range of 5° and 100°. Scanning electron microscopy (SEM; SUPRA 55, Carl Zeiss NTS GMBH) was performed to assess the surface morphology of the CSNPs. Transmission electron microscopy (TEM) was performed to visualize the size and shape of the CSNPs, and it was performed on a Cu grid containing carbon coating.

2.3 Batch adsorption studies

The adsorption of Zn²⁺ ions by CSNPs was performed in batch adsorption by varying the independent variables. The optimization of independent variables was performed using the central composite design (CCD) of response surface methodology (RSM). All the experiments were performed for 50 mL test solutions in an orbital shaker at 100 rpm. A full factorial design with 20 experiments was used to make it efficient for fitting data and building models, and important factors and their details are given in Table 1. The construction of CCD and building of models were performed with Design Expert 13 software.

The adsorption efficiency and loading capacity of CSNPs were calculated using the following equations:

2.4 Antimicrobial assay

The antibacterial properties of CSNPs were tested using the agar well diffusion method on Mueller-Hinton Agar (MHA) medium. Sterilized MHA was poured into sterile Petri dishes under aseptic conditions and left to solidify for 1 h at room temperature. Overnight cultures of E. coli (Gram-negative) and S. aureus (Gram-positive) were standardized to approximately 10⁸ CFU/mL, matching the 0.5 McFarland standard, and evenly spread over the MHA surface using sterile cotton swabs. Sterile wells, 7–8 mm in diameter, were created in the agar using a cork borer. CSNP suspensions were prepared in sterile distilled water, and 50 µL of each suspension was added to the wells. The plates were kept at room temperature for 30 min to allow diffusion of the NPs into the agar and then incubated at 37°C for 24 h to facilitate bacterial growth and interaction with the NPs. After incubation, the zones of inhibition (ZOIs) around the wells were measured with a calibrated ruler to evaluate the CSNPs’ antibacterial effectiveness. Each experiment was performed in triplicate to ensure reproducibility and statistical reliability.

2.5 Diet preparation and experimental design

Fingerlings of O. niloticus (L.) were obtained from nursery ponds and acclimatized to laboratory conditions in an indoor spun-glass aquarium for 2 weeks. The aquarium was aerated continuously, and a controlled light source maintained a 12-h light–dark cycle. A total of 30 fish, with an average weight of 16.21 ± 0.24 g, were randomly distributed among three experimental groups, with 10 fish housed in each 3 L aquarium tank, replicated four times. The groups included Experiment 1, where fish were fed a commercially available feed; Experiment 2, serving as the control group; and Experiment 3, where fish were provided with a CS-enriched feed. All aquariums were equipped with compressed air pumps to ensure proper aeration. The fish were fed their respective diets twice daily, at 9:00 AM and 2:00 PM, for 45 days, with feed provided until the fish were satiated. Each day, settled waste and half of aquarium water were removed and replaced with fresh, aerated tap water from a holding tank. Mortality records were maintained daily, and any deceased fish were promptly removed from the tanks to ensure optimal experimental conditions.

2.6 CSNPs loaded with fish feed

The synthesized CSNP pellets were dispersed in distilled water to prepare a uniform 1% solution that provides moderate concentration of CSNPs. This suspension was loaded into a 20 mL syringe and sprayed evenly onto the prepared fish feed. The CS-coated feed was thoroughly mixed and then dried in a hot air oven at 100°C. Once dried, the CS-enriched feeds (as described in Table 2) were provided to the fish in experimental tanks 1–3 for a duration of 1 week. At the end of the observation period, the fish were weighed, and their lengths were measured to evaluate their growth. The initial and final weights, as well as the initial and final lengths of the fish, were recorded to assess the impact of the CS-loaded feed on their growth and development.

3.1 Synthesis of CSNPs

The ionic gelation approach was used in the current study to create CS nanosuspension. The above-mentioned procedures are advantageous for CSNP synthesis since they are quick and gentle, requiring no organic solvents or high temperatures. It hinges on the ionic connection between the positively and negatively loaded amino groups in the acidic CS solution and the aqueous STPP solution. The molecules with opposing charges can form inter-molecular crosslinks due to the ongoing magnetic churning. TPP was gradually included into the CS solution to produce the ionotropic gelation of CSNPs.

The synthesized CSNPs had monodispersed NPs of around 100 nm in size and consistently low polydispersity index (PDI) values. The stability and structural uniformity of NPs were assessed using the PDI. In this study, low PDI values denote higher levels of monodisperse CSNPs and higher levels of particle equilibrium, whereas higher values suggest lower levels of particle equilibrium and CSNP accumulation. Opacity of the synthesized CSNPs was verified by the presence of opalescence, which was evaluated for physico-chemical traits like crystalline structure, thermal potential, ultra-structural morphology, and the dimension of particles.

3.2 Characterization of CSNPs

3.2.1 UV–vis absorption spectroscopy

The optical properties of CSNPs were analyzed using UV–visible spectroscopy within a wavelength range of 200–500 nm. A sharp absorption peak was observed at 240 nm in the UV–vis spectra, which is due to n–σ^* of the –NH₂ and –OH groups (Figure 1). The band around 280 nm is due to the n–π^* of the acetyl group of CS. The bands observed in CS bulk as well as minor shifts in wavenumbers due to the nanosize nature of CS confirm the formation of CSNPs. Similar observations were reported for the biologically synthesized CSNPs [15].

Figure 1

UV–vis spectra of CSNPs prepared via the ionic gelation method (data from authors’ own research).

3.2.2 FT-IR spectroscopy analysis

The molecular structures of CSNPs were analyzed using FT-IR spectroscopy, as shown in Figure 2. The FT-IR spectra revealed characteristic peaks, indicating specific molecular vibrations and functional groups. A broad absorption band at 3,358 cm⁻¹ corresponds to the –OH groups of the CSNPs, and a small peak at 3,252 cm⁻¹ overlapped with the –OH peak indicates the –NH stretching. The peak at 2,873 cm⁻¹ is due to the symmetrical stretching of –CH groups [16]. Bands at 1,647 and 1,587 cm⁻¹ correspond to the amide I C═O stretching and amide II C–N stretching vibrations, respectively, and these bands were shifted compared to the CS bulk, and this is due to the formation of NPs and their interactions with the cross linker TPP used in this study [17,18]. The peak at 1,145 cm⁻¹ corresponds to the glycosidic linkage group of CSNPs. The peak at 695 cm⁻¹ is primarily due to N–H bending that arises due to NH₃ ⁺ in the ionic gelation method. These findings confirm the presence of various functional groups, validating the molecular structure and composition of the synthesized CSNPs.

Figure 2

FT-IR spectrum of CSNPs prepared via the ionic gelation method. Inset: FT-IR spectrum of CS (data from authors’ own research).

3.2.3 Field emission SEM analysis

SEM was employed to analyze the microstructures of the synthesized CSNPs, focusing on their size, shape, and dispersion patterns (Figure 3). The SEM images revealed that the CSNPs exhibited a spherical shape with a homogeneous nanostructure. The particles were uniformly distributed and of nanoscale size, indicating that the interaction between CS and TPP molecules successfully facilitated the formation of well-defined CSNPs. The observed morphology aligns with the findings from previous studies, confirming the reliability of the synthesis method in producing consistent particle structures [19,20]. These results highlight the potential of CSNPs for diverse applications across various fields, including tissue engineering, environmental remediation, biomedical imaging, and drug delivery systems. This consistent particle morphology provides a strong foundation for exploring the functional capabilities of CSNPs in these domains.

Figure 3

SEM images of CSNPs prepared via the ionic gelation method (data from authors’ own research).

3.2.4 High-resolution TEM (HR-TEM) analysis

HR-TEM analysis confirmed the size and microscopic structure of the synthesized CSNPs, prepared under optimal conditions (Figure 4). The NPs were spherical, with sizes ranging from 10 to 40 nm. HR-TEM images revealed well-defined lattice fringes, indicative of a high degree of crystallinity in the samples [21]. The precise dry size of the CSNPs was measured through this technique, providing detailed structural insights. These findings highlight the strong interaction dynamics between CS and TPP, which play a crucial role in the formation and stability of CSNPs.

Figure 4

TEM image of CSNPs prepared via the ionic gelation method (data from authors’ own research).

3.2.5 XRD measurement

XRD analysis was utilized to assess the morphological characteristics and crystallite dimensions of CSNPs. The XRD patterns of the CSNPs are presented in Figure 5. The crystalline quality of the CSNPs was demonstrated by a prominent diffraction peak at a 2θ range of 10° and 20°. The small and less intense peak at 10° is due to the hydrated crystalline structure of the CSNPs, confirming the reduction in crystallinity due to nanoscale compared to the bulk CS [22]. The intense peak around 20° is due to the crystalline structure of CS, showcasing the regular arrangement of the polymeric chains [23]. The presence of a broader band further indicated an imperfect crystal structure, likely due to the synthesis and processing conditions, especially the use of TPP. These findings provide insight into the crystalline properties and structural behavior of CSNPs.

Figure 5

XRD patterns of CSNPs prepared via the ionic gelation method. Inset: CS XRD pattern (data from authors’ own research).

3.3 RSM-based optimization

CCD was utilized to optimize the adsorption of Zn²⁺ ions using CSNPs. The study focused on three independent variables: pH, contact time, and initial Zn²⁺ concentration. The CCD included 20 experimental runs, consisting of 6 axial points, 6 central points, and 8 cubic points distributed within the design space. Tables 3 and 4 provide the experimental and predicted design matrices as well as the analysis of variance (ANOVA) results for the Zn²⁺ removal process.

Design-Expert 13 software was employed to develop a quadratic model, yielding a second-order polynomial equation (equation (3)) that defines the relationship between the selected factors and the Zn²⁺ removal efficiency, given by

The influence of each factor (pH, contact time, and concentration) on Zn²⁺ adsorption by CSNPs is reflected in the coefficients of the quadratic equation (equation (3)). Positive coefficients indicate that increasing the factor level enhances adsorption, whereas negative coefficients suggest a reduction in the adsorption efficiency. The equation also accounts for interactions between factors (two-factor terms) and the impact of these interactions on adsorption efficiency (curvature terms). The strong correlation between predicted and experimental results, as shown in Table 3, confirms the validity of the selected model and demonstrates improved Zn²⁺ adsorption in the experiments.

The ANOVA results in Table 4 affirm the robustness of the model for predicting Zn²⁺ removal using CSNPs. The extremely small p-value (<0.0001) highlights the model’s statistical significance, indicating the results are highly unlikely due to random variation. A high F-value of 92.77 further reinforces the model’s reliability, as larger F-values suggest a very low probability (0.01%) of the findings occurring by chance [24]. Moreover, the lack-of-fit F-value exceeding 0.1 indicates minimal unexplained variation within the model. The alignment between predicted and actual values was assessed for accuracy, as shown in Figure 6d. The points are randomly distributed along a straight line, demonstrating a normal data distribution without significant deviations, eliminating the need for data transformation and validating the model. Additionally, correlation coefficients nearing 1 (0.988), along with high adjusted and predicted R-squared values (0.977 and 0.903, respectively), further corroborate the model’s effectiveness [25]. An adequate precision value of 28.3 underscores the model’s strong predictive capability.

Figure 6

3D surface plots of independent variable interactions: (a) pH vs contact time, (b) pH vs initial concentration, (c) initial concentration vs contact time, and (d) predicted vs actual values (data from authors’ own research).

The interaction between independent variables and their impact on the binding of Zn²⁺ ions by CSNPs can be effectively visualized using 3D surface plots, as shown in Figure 6. These plots provide critical insights into the adsorption process. Figure 6a illustrates the combined effect of pH and contact time on Zn²⁺ ion binding. It reveals that as the pH and contact time increase, the binding efficiency also improves, reaching its peak at pH 7. However, beyond pH 7, the efficiency declines, even with extended contact times. At lower pH levels, competitive adsorption by hydronium ions on the active surface sites results in reduced binding efficiency. As the pH increases, this competition diminishes, enabling better adsorption of Zn²⁺ ions. The improvement in efficiency with longer contact times is attributed to the increased opportunity for Zn²⁺ ions to interact with the CSNP surface. The 3D plot clearly demonstrates that both pH and contact time significantly influence adsorption efficiency.

Figure 6b illustrates the interaction between pH and initial concentration. At the optimal pH of 6, the removal efficiency decreases with an increase in initial Zn²⁺ concentration. This decline occurs because the surface active sites on CSNPs become saturated as the concentration increases, reducing the percentage of ions bound. Figure 6c shows the synergistic effect of initial concentration and contact time on Zn²⁺ ion adsorption. While prolonged contact time enhances the adsorption efficiency, increasing the initial concentration diminishes it due to limited availability of active sites.

3.4 Antimicrobial investigation of CSNPs

The antimicrobial activity of the CSNPs synthesized in this study was evaluated against E. coli and S. aureus, with the results summarized in Table 5. The findings indicate that CSNPs demonstrated significantly superior antimicrobial activity with 25 and 24 mm of ZoI against both E. coli and S. aureus compared to the standard antibiotic streptomycin at 20 µL concentration. This enhanced activity at low concentrations can be attributed to the interaction of the NPs with the microbial cell membrane, which leads to membrane disruption and leakage of intracellular contents, ultimately compromising the viability of the microbes [9,11].

3.5 Growth and feed conversion efficiency of O. niloticus

Table 6 highlights that the CSNP-formulated feed significantly enhanced the growth performance of O. niloticus compared to both commercial and control basal feeds. Fish fed with the CSNP-formulated diet exhibited a higher feed intake (40.43 ± 0.75) compared to those on the control feed. Throughout the experimental period, the CSNP-supplemented group demonstrated excellent health and growth efficiency, with a 100% survival rate, consistent across all diet groups, including the control and commercial feeds. The improved growth performance was attributed to better intestinal villi development, enhanced nutrient absorption, increased specific growth rate (SGR), and higher feed consumption in the CSNP-formulated diet group [26]. These factors collectively contributed to the observed superior growth in O. niloticus. Additionally, the dietary supplementation with CS-enriched feed improved the feed conversion efficiency and overall productivity, consistent with similar findings reported for Dicentrarchus labrax [27]. This demonstrates the efficacy of CSNPs as a dietary supplement for enhancing fish growth and health.

3.6 Hematological analysis

The red blood cell (RBC) and white blood cell (WBC) counts in the control group increased gradually from the first to the third week but showed a significant rise after being fed with an enriched diet formulated in this study starting from the first week. The RBC levels improved notably due to the loaded CSNP diet. While the control group showed gradual improvement, fish treated with the CSNPs/kg diet exhibited a substantial boost in RBC and WBC levels of 20.0 × 10⁶/µL and 3.2 × 10³/µL, respectively (Figure S1). WBCs are an essential component of innate immunity in fish and release humoral chemicals like cationic antimicrobial peptides, complement system components, lectins, and cytokines [28]. The higher WBC count exhibited by fish fed with CSNP diet suggests the improved immunity compared to other diets, and this can be attributed to the enhanced activity and proliferation of lymphocytes and macrophages. Further, the cytokine production may be stimulated that promotes immune cell activation. The stress reduction and nutritional enhancement of CSNPs improved the RBC production compared to other diets supplied to the fish. Similar results were reported for adding the nanochitosan/zeolite composite to rainbow trout’s diet, which resulted in an increase in serum protein levels [29].

3.7 Future directions

The findings of this study highlight the potential of CSNPs for addressing Zn²⁺ ion contamination in water in a sustainable manner. Furthermore, Zn²⁺ ion-bound CSNPs can be evaluated for their antimicrobial properties, leveraging the synergistic effects of Zn²⁺ ions and CSNPs. The incorporation of Zn²⁺ ion-bound CSNPs into aquatic environments can reduce pathogen loads, improving fish health by minimizing infections. The application of CS formulations in fish feed has already shown benefits, such as promoting healthier growth in O. niloticus. The inclusion of Zn²⁺ ion-bound CSNPs in feed formulations can further enhance the production and activation of immune cells, stimulate macrophage activity, and increase the resilience of O. niloticus to diseases and environmental stress. However, optimizing the Zn²⁺ ion loading onto CSNPs is critical to prevent zinc toxicity and ensure a balanced approach to fish health management. Current investigations are underway to determine the optimal Zn²⁺ ion loading levels that contribute to the overall growth and health of O. niloticus, paving the way for a sustainable and effective practice.