MATERIALS AND METHODS

Ethical approval

All procedures involving experimental animals were reviewed and approved by the Chulalongkorn University Animal Care and Use Committee, Chulalongkorn University, Bangkok, Thailand (Approval No. 2431095). All experiments were conducted in accordance with institutional animal welfare guidelines and applicable regulatory standards. Appropriate measures were implemented throughout the study to minimize stress and discomfort to the fish. During challenge experiments, fish exhibiting severe loss of equilibrium, inability to feed, or unresponsiveness were considered moribund and were humanely euthanized in accordance with approved ethical guidelines.

Study period and location

The study was conducted from May to August 2025 at the Center of Excellence in Wildlife, Exotic, and Aquatic Animal Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand. Physicochemical characterization and biological evaluations of CUR-NLCs, including antibacterial assays, feeding trials, and challenge experiments, were carried out under controlled laboratory conditions. The feeding experiment was conducted over 60 days, followed by a 15-day post-challenge observation period.

Study design

Prior to the feeding trial, Nile tilapia fingerlings (n = 600; initial body weight, 6-8 g) were treated prophylactically with 50 ppm formalin and acclimatized for 14 days in fiberglass tanks with a working volume of 150 L supplied with continuously aerated, flow-through dechlorinated tap water.

Following acclimatization, fish were randomly allocated to four dietary treatments comprising a basal control diet, free CUR, CUR-NLC, and blank NLC. Each treatment was performed in triplicate, with 50 fish stocked per tank.

Throughout the 60-day feeding period, environmental conditions were monitored daily to ensure stable culture conditions. Water temperature ranged from 26°C to 28°C, dissolved oxygen concentrations ranged from 5.34 to 5.78 mg/L, and pH values ranged from 7.38 to 8.20. Approximately 30%-50% of the water volume was exchanged daily to maintain suitable water quality. Fish were fed their respective diets twice daily at a feeding rate corresponding to 3% of total body weight.

Blinding was not performed during feed preparation, feeding, sampling, or challenge procedures because the dietary treatments were visibly distinguishable.

Chemicals

CUR (95% purity, high-performance liquid chromatography grade; Batch No. NIS21070222-TMR-01) was obtained from Primo Trading Co., Ltd. (Bangkok, Thailand). Lipid excipients, including sorbitan oleate, cetearyl alcohol, cocoglucoside, polyoxyethylene (20) sorbitan monolaurate (Tween 20), poloxamer 188, and glycerol, were purchased from Croda (Thailand) Co., Ltd. (Bangkok, Thailand). Ethoxydiglycol was supplied by Myskin Recipe Co., Ltd. (Bangkok, Thailand). All other chemicals and reagents used in this study were of analytical grade and suitable for experimental and biological applications.

Formulation of CUR-NLCs

CUR-NLCs were prepared using a phase-inversion compositional approach coupled with high-energy homogenization. This formulation strategy was selected to facilitate stable incorporation and sustained dietary delivery of CUR, whose low aqueous solubility and chemical instability limit its effectiveness in conventional feed formulations. The preparation protocol followed a previously validated NLC platform [40], and formulation parameters were maintained to enable direct evaluation of compound-specific biological effects rather than differences arising from carrier architecture.

Briefly, 200 mg of CUR was incorporated into a lipid phase comprising a solid lipid component (cetearyl alcohol-cocoglucoside blend, 1.0 g), a liquid lipid component (sorbitan oleate, 3.0 g), and ethoxydiglycol (3.0 g), and the mixture was continuously agitated at 60°C to ensure complete solubilization. Accordingly, the formulation contained an approximate solid-to-liquid lipid ratio of 1:3. Separately, an aqueous phase containing polyoxyethylene (20) sorbitan monolaurate (3.0 g), poloxamer 188 (2.0 g), and glycerol (2.5 g) was prepared in deionized water and maintained at the same temperature. The heated aqueous phase was gradually added to the lipid mixture and homogenized at 500 rpm for 5 min to obtain a preliminary dispersion. Subsequently, ultrasonic processing was performed at 30% power output for 5 min using intermittent 30-s cycles with resting intervals to avoid excessive heat generation. Following sonication, the CUR-NLC formulation was allowed to equilibrate to ambient temperature before further physicochemical and biological characterization.

Physicochemical characterization of CUR-NLCs

The physicochemical characteristics of CUR-NLCs, including particle size, size distribution, and surface charge, were determined using dynamic light scattering with a Zetasizer Nano ZS system (Malvern Instruments, Malvern, UK). Samples were diluted 50-fold with deionized water before analysis to minimize light-scattering interference. All measurements were performed at 25°C, and each parameter was measured in triplicate.

Nanoparticle morphology was examined using transmission electron microscopy (HT7800; Hitachi High-Tech Corporation, Tokyo, Japan) operated at 80 kV [44]. For sample preparation, CUR-NLC dispersions were diluted (1:50) with deionized water adjusted to pH 7.0 and deposited onto carbon-coated copper grids. Excess liquid was removed using filter paper, and the grids were stained with uranyl acetate staining solution for 2 min, rinsed with deionized water, air-dried, and subsequently examined microscopically. Transmission electron microscopy analysis was used for qualitative morphological assessment.

Encapsulation capacity and release characteristics of CUR-NLCs

The proportion of CUR successfully incorporated into NLCs was evaluated and compared with free CUR preparations using centrifugal separation based on molecular size exclusion. Regenerated cellulose ultrafiltration devices with a nominal cutoff of 30 kDa (Amicon Ultra-15, Merck Millipore Ltd., Burlington, MA, USA) were used in accordance with a validated analytical procedure [40]. For each formulation, 1.5 mL was transferred to filtration units and centrifuged according to the manufacturer's instructions, thereby separating carrier-associated CUR from the freely dissolved fraction. The filtrate containing unassociated CUR was recovered by solvent extraction as previously described by [41], passed through a 0.2 μm nylon membrane, and quantified by high-performance liquid chromatography.

Quantitative determination of CUR was performed using a Waters liquid chromatography system equipped with a photodiode array detector (Model 2998; Waters Corporation, Milford, MA, USA). Separation was achieved on a reversed-phase C18 column (Ascentis, 5 μm, 250 × 4.6 mm; Sigma-Aldrich, St. Louis, MO, USA). The mobile phase consisted of acetonitrile and methanol (60:40, v/v) delivered at a flow rate of 1.0 mL/min. The sample volume was 20 μL, and detection was performed at 240 nm. Each chromatographic run was completed within 15 min.

Encapsulation efficiency (EE) was calculated using the following equation:

\documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} \[ EE(\%)=\frac{\left({C}_{i},{C}_{f}\right)}{{C}_{i}}\times 100 \] \end{document}

where \documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} ({C}{i}) \end{document}represents the initial CUR concentration and \documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} ({C}{f}) \end{document}corresponds to the amount of CUR detected in the filtrate.

The loading capacity (LC), which describes the proportion of CUR entrapped relative to the total lipid mass, was calculated as follows:

\documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} \[ LC(\%)=\frac{\left({C}_{i},{C}_{f}\right)}{Total lipid mass}\times 100 \] \end{document}

The release profile of CUR from CUR-NLCs was investigated under simulated physiological conditions using a diffusion-controlled dialysis technique. Briefly, 1.5 mL of CUR-NLC dispersion was transferred into dialysis membranes (molecular weight cutoff 3.5 kDa; Merck Millipore) and immersed in 30 mL of phosphate-buffered saline (pH 6.8) containing ethanol (95:5, v/v) to maintain sink conditions [40]. The system was incubated at 28°C in an orbital shaking incubator (Vision Scientific Co., Daejeon, South Korea) operating at 200 rpm.

At predetermined intervals (0-48 h), aliquots were withdrawn from the release medium and replaced with fresh buffer to maintain a constant volume. The released CUR concentration at each time point was determined using high-performance liquid chromatography. Release kinetics were analyzed using the Avrami model:

\documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} \[ R=1-exp⁡[-(kt{)}^{n}] \] \end{document}

where \documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} (R) \end{document}denotes cumulative release at time \documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} (t) \end{document}, \documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} (k) \end{document}is the apparent release rate constant, and \documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} (n) \end{document}represents the release mechanism.

Functional group analysis

To investigate molecular interactions between CUR and the lipid matrix, Fourier-transform infrared spectroscopy was performed [45]. Spectra of free CUR and CUR-NLCs were obtained using a Nicolet Summit Pro Fourier-transform infrared spectrometer (Thermo Scientific, Waltham, MA, USA). Spectra were recorded over the range of 500-4000 cm⁻¹ at a resolution of 4 cm⁻¹.

Samples were prepared by mixing formulations with potassium bromide at a 1:100 ratio, followed by grinding and pellet formation. Each spectrum was generated from 64 accumulated scans, and all analyses were performed in triplicate to ensure reproducibility.

Evaluation of antibacterial activity

The antibacterial activity of free CUR and CUR-NLCs was evaluated against three S. agalactiae isolates (FNA07, FPrA02, and ENC06) obtained from diseased Nile tilapia in Thailand. The assay was performed using the agar diffusion method. All analyses were conducted in triplicate for each isolate and formulation.

Bacterial isolates were cultured on tryptic soy agar supplemented with 5% sheep blood and incubated at 28°C for 24 h [46]. Fresh colonies were suspended in sterile 0.85% saline and adjusted to a turbidity equivalent to a 0.5 McFarland standard. Bacterial suspensions were evenly spread onto Mueller-Hinton agar supplemented with 5% sheep blood. Wells measuring 6 mm in diameter were prepared aseptically, and 50 μL of either free CUR or CUR-NLC formulation was added to each well. Plates were incubated at 28°C for an additional 24 h, and antibacterial activity was determined by measuring the diameters of growth inhibition zones (IZ) with a calibrated Vernier caliper to a precision of ±0.02 mm.

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

The inhibitory and bactericidal activities of CUR and CUR-NLCs were evaluated using the broth dilution method [44]. Stock formulations were serially diluted twofold in Mueller-Hinton broth to obtain concentrations ranging from 2,000 to 3.9 ppm. Aliquots were transferred into sterile 96-well microplates, and each well received 100 μL of bacterial suspension adjusted to a final concentration of 1 × 10⁶ colony-forming units/mL.

Following inoculation, plates were incubated aerobically at 28°C for 24 h. The MIC was defined as the lowest concentration that completely inhibited visible bacterial growth. To determine the MBC, aliquots from wells without visible turbidity were plated onto Mueller-Hinton agar and incubated under identical conditions. The MBC was defined as the lowest concentration at which no bacterial colonies were recovered.

Preparation of experimental feed

Commercial tilapia pellets (CP-7710) were used as the basal diet (moisture, 7.6%; crude protein, 30.5%; crude lipid, 6.5%; and ash, 8.2%, according to the manufacturer's specifications) and were externally coated with CUR, CUR-NLCs, or blank NLC formulations. Because the coating formulations were applied at relatively low inclusion levels relative to the total feed mass, substantial alteration of the proximate nutritional composition of the commercial basal diet was not expected.

Coating solutions were prepared at a final concentration of 2 mg/mL and applied at a rate of 1 mL/g feed [45], resulting in an approximate CUR inclusion level of 2000 mg/kg feed. The final dietary CUR concentration was calculated from the formulation concentration and the coating volume applied per unit of feed. Analytical verification of CUR retention in coated pellets after drying and storage was not performed in the present study. However, the coating procedure was standardized across all experimental diets to ensure consistency among treatments. This supplementation level was selected based on previously reported dietary CUR inclusion levels used in Nile tilapia and other fish species [27, 47].

The formulated NLC dispersions were homogeneous, low-viscosity fluids at ambient temperature, facilitating uniform distribution over the commercial feed pellets. Pellets were gently agitated using a sterile stainless-steel spatula to ensure uniform surface coating. Control diets were prepared using distilled water in place of active formulations. Following coating, the feeds were dried overnight at room temperature(28–30°C) and subsequently stored at 4°C until use.

During the feeding period, fish readily accepted all experimental diets, including pellets coated with CUR and CUR-NLC formulations, without observable feed rejection or abnormal feeding behavior. In addition, no visible discoloration of the rearing water or apparent residue associated with CUR or CUR-NLC leaching was observed during feeding, indicating acceptable coating adherence and physical stability of the supplemented pellets under the experimental conditions.

Evaluation of growth performance and feed utilization

Fish growth performance and feed utilization efficiency were evaluated on days 30 and 60 of the feeding trial. The variables assessed included Weight gain (WG), Specific growth rate (SGR), mean daily intake (MDI), feed conversion ratio (FCR), protein efficiency ratio (PER), and hepatosomatic index (HSI). These parameters were calculated using established methods in aquaculture nutrition studies [48].

WG (g/fish) was calculated as follows:

\documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} \[ WG=FW-IW \] \end{document}

where FW and IW represent final and initial body weight, respectively.

SGR (%/day) was calculated as:

\documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} \[ SGR=100\times \frac{ln⁡FW-ln⁡IW}{T} \] \end{document}

where T denotes the duration of the feeding period.

MDI (g/fish/day) was determined as:

\documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} \[ Mean daily intake=\frac{Total feed intake/T}{Number of fish per tank} \] \end{document}

FCR was calculated as:

\documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} \[ FCR=\frac{Feed intake}{Wet weight gain} \] \end{document}

PER was calculated using the following equation:

\documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} \[ PER=\frac{Wet weight gain}{Protein intake} \] \end{document}

HSI (%) was calculated as:

\documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} \[ HSI=\frac{Liver weight}{Body weight}\times 100 \] \end{document}

Length–weight relationship, growth pattern, and relative condition factor (Kn)

At the end of the 60-day feeding period, body size scaling and physiological condition were evaluated. The relationship between body weight and total length was determined using the allometric equation described by Pauly [49]:

\documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} \[ W=a{L}^{b} \] \end{document}

where W denotes body weight (g), L denotes total length (cm), and a and b are the intercept and slope of the regression model, respectively.

To estimate these parameters, the equation was transformed into logarithmic form:

\documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} \[ log⁡W=log⁡a+blog⁡L \] \end{document}

Growth patterns were interpreted according to the value of b. A value of b = 3 indicated isometric growth, whereas values greater than or less than 3 represented positive and negative allometric growth, respectively [50].

The relative condition factor (Kn) was further evaluated according to the method described by Le Cren [51]:

\documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} \[ Kn=\frac{{W}_{o}}{{W}_{c}} \] \end{document}

where \documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} ({W}{o}) \end{document}is the observed body weight and \documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} ({W}{c}) \end{document} is the predicted body weight estimated from the population-derived length-weight equation \documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} (\left(W,a{L}^{b}\right)) \end{document}. The predictive equation was generated using pooled measurements from all fish sampled on day 60. Values of Kn >1 indicated relatively better physiological condition, whereas values <1 indicated poorer condition.

Disease challenge and survival analysis

For the challenge experiment, the S. agalactiae isolate ENC06 was selected because it exhibited greater susceptibility to CUR-NLC treatment during antibacterial screening and has been repeatedly associated with severe streptococcosis outbreaks in Nile tilapia farms in Thailand [52, 53].

At the end of the feeding trial, 30 fish from each dietary treatment were randomly selected for the challenge study. Before inoculation, fish were anesthetized with clove oil at a concentration of 20 mg/L to minimize handling stress and ensure humane treatment [54].

Experimental infection was performed by intraperitoneal injection of 100 μL bacterial suspension containing 1 × 10⁶ colony-forming units/mL of S. agalactiae ENC06. The challenge dose was selected based on a preliminary determination of the median lethal dose using the same strain under identical experimental conditions, to establish a sublethal dose appropriate for survival analysis.

Following inoculation, fish were transferred immediately to well-aerated recovery tanks, and handling time was minimized. Mortality was monitored daily during the 15-day post-challenge period. Confirmation of infection was achieved through re-isolation of S. agalactiae from kidney, spleen, and brain tissues of moribund and freshly dead fish.

During the challenge period, water temperature, pH, and dissolved oxygen remained within optimal ranges at 29.9 ± 0.05°C, 7.37 ± 0.06, and 4.97 ± 0.04 mg/L, respectively.

Host resistance was evaluated using cumulative mortality and relative percent survival (RPS) according to the method described by Amend [55]:

\documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} \[ \begin{aligned} Cumulative mortality (\%)=\frac{Number of dead fish}{Initial number of fish}\times 100 \\ RPS(\%)=\left[1,\frac{Mortality percentage in treated group}{Mortality percentage in control group}\right]\times 100 \end{aligned} \] \end{document}

Statistical analysis

Data were analyzed using SPSS Statistics version 29 (IBM Corp., Armonk, NY, USA). Before analysis, the normality of the residuals was assessed using the Shapiro-Wilk test, and the homogeneity of variance was evaluated using Levene's test.

The tank was considered the experimental unit, whereas measurements from individual fish were treated as subsamples and averaged before analysis. Parameters measured at days 30 and 60 were analyzed using repeated-measures analysis of variance, with time considered the within-subject factor and dietary treatment considered the between-subject factor.

Variables measured at a single time point were analyzed using one-way analysis of variance followed by Tukey's honestly significant difference post hoc test. Survival responses were analyzed using Kaplan-Meier survival curves, and group differences were evaluated using the log-rank test and Cox proportional hazards regression analysis.

Relationships among nanoparticle physicochemical characteristics, antibacterial activity, growth-related variables, and disease resistance parameters were investigated using Pearson's correlation analysis. Statistical significance was accepted at p < 0.05.