1. INTRODUCTION:

Fucoxanthin (FUCO) is a marine xanthophyll carotenoid known for its promising pharmacological effects, including antioxidant, antitumor, anti-inflammatory, and antidiabetic activities^1,2. However, its practical application is limited by hydrophobicity, chemical instability, and poor intestinal absorption^2,3. These limitations greatly restrict its therapeutic potential despite extensive in vitro and preclinical evidence.

Consequently, nanoscale delivery systems have been increasingly employed to overcome solubility and stability challenges. Biopolymers such as chitosan (CS) and alginate (ALG) have demonstrated excellent biocompatibility and gastrointestinal mucoadhesiveness^4,5. However, the poor aqueous solubility of native CS under physiological conditions limits its application in oral formulations^4,5. To address this limitation, chitosan oligosaccharide (CSO), a depolymerized and water-soluble derivative of CS, has been utilized owing to its enhanced aqueous solubility and mucosal permeability. Furthermore, CSO interacts strongly with negatively charged biopolymers such as ALG, facilitating the formation of stable polyelectrolyte complexes exhibiting pH-responsive behavior^4,6. These features are particularly advantageous for developing oral drug delivery systems capable of withstanding the harsh gastric environment and releasing their payloads in the intestine⁷. In this study, we developed and optimized fucoxanthin-loaded nanoparticles composed of CSO and ALG (FUCO-COANPs) using a Box–Behnken design. Their physicochemical characteristics, release kinetics, in vitro bioaccessibility, and functional bioactivities were systematically evaluated to assess their potential for enhancing FUCO oral delivery.

2. MATERIALS AND METHODS:

2.1. Materials:

Fucoxanthin (FUCO, > 98% purity) was obtained from Xi’an Sgonek Biotech Co., Ltd. (China). Chitosan oligosaccharide (CSO, MW 1500–1600 g/mol, deacetylation > 98%) was purchased from Kono Chem Co., Ltd. (China). Sodium alginate (ALG, MW 8.0 × 10⁴–1.2 × 10⁵ g/mol) was supplied by Sigma-Aldrich (USA). Tween™ 80 and other analytical-grade reagents were obtained from Acros Organics (Thermo Fisher Scientific, Belgium). Calcium chloride and organic solvents were sourced from Carlo Erba Reagents (France). Ultrapure water was prepared using a Barnstead MicroPure system. All chemicals were of analytical grade and used without further purification.

2.2. Fabrication of FUCO-COANPs:

FUCO-COANPs were fabricated using an emulsification–ionic gelation method, modified from the protocol of Shamekhi et al.⁸ to enhance formulation efficiency. Briefly, 1 mL of FUCO dissolved in ethanol was gradually added to 20 mL of 0.6 mg/mL ALG solution containing a predetermined volume of Tween™ 80 and stirred at 1000 rpm for 10 min at room temperature. Gelation was induced by adding 4 mL of 0.67 mg/mL CaCl₂ solution, followed by continuous stirring for another 30 min. The emulsion was sonicated for 15 min to reduce particle size. Subsequently, 4 mL of CSO dissolved in 1% (v/v) acetic acid was introduced, followed by an additional 30 min of stirring to complete nanoparticle formation. The resulting dispersion was stored overnight in the dark before characterization.

2.3. Design and optimization of FUCO-COANPs:

The FUCO-COANP formulation was optimized using a Box–Behnken design (BBD) involving three independent variables at three coded levels. The experimental design and statistical analyses were performed using Design-Expert^® software (Stat-Ease Inc., Minneapolis, USA). The selected variables were the CSO:ALG mass ratio (X₁), Tween™ 80 concentration (X₂), and FUCO concentration (X₃), while particle size (PS, Y₁) and encapsulation efficiency (EE, Y₂) were designated as the responses. Fifteen experimental runs were conducted according to the design matrix. The factor levels and response criteria are presented in Table 1.

Table 1: Formulation factors and response variables selected in the Box–Behnken design for the development of FUCO-COANPs.

Factors	Levels used
Factors	Low	Medium	High
X₁ = CSO:ALG mass ratio	0.04:1	0.08:1	0.12:1
X₂= Tween™ 80 (% w/v)	1.5	2	2.5
X₃ = FUCO (mg/mL)	4	6	8
Responses	Constraints
Y₁ = PS (nm)	Minimize
Y₂ = EE (%)	Maximize

Table 2: Experimental results from the Box–Behnken design for FUCO-COANPs.

Run	Factors			Responses
Run	X₁	X₂	X₃	Y₁	Y₂
1	0.04:1	1.5	6	258 ± 25	64.2 ± 2.3
2	0.12:1	1.5	6	357 ± 19	72.3 ± 1.9
3	0.04:1	2.5	6	265 ± 15	74.7 ± 0.8
4	0.12:1	2.5	6	357 ± 23	87.2 ± 1.1
5	0.04:1	2.0	4	384 ± 31	63.8 ± 1.9
6	0.12:1	2.0	4	457 ± 29	84.1 ± 2.1
7	0.04:1	2.0	8	366 ± 18	67.7 ± 0.5
8	0.12:1	2.0	8	479 ± 34	83.5 ± 1.8
9	0.08:1	1.5	4	423 ± 17	65.2 ± 2.3
10	0.08:1	2.5	4	334 ± 26	78.7 ± 1.6
11	0.08:1	1.5	8	318 ± 29	73.4 ± 0.9
12	0.08:1	2.5	8	436 ± 14	79.6 ± 1.2
13*	0.08:1	2.0	6	368 ± 22	74.2 ± 0.7
14*	0.08:1	2.0	6	346 ± 13	75.8 ± 1.4
15*	0.08:1	2.0	6	357 ± 28	72.6 ± 0.9

* Runs 13–15 represent the center point conditions in the Box–Behnken experimental matrix.

2.4. Characterization:

The physicochemical properties of FUCO-COANPs, including particle size (PS), polydispersity index (PDI), and zeta potential (ZP), were measured by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, UK) at ambient temperature⁹. Transmission electron microscopy (TEM; JEOL Ltd., Japan) was used to observe nanoparticle morphology⁹. Encapsulation efficiency (EE) was determined by quantifying the amount of unencapsulated FUCO remaining in the supernatant after ultracentrifugation at 35,000 rpm for 45 min at 4 °C. The absorbance of the supernatant was measured at 450 nm using a UV–Vis spectrophotometer (Agilent Technologies, Germany). EE (%) was calculated using Equation (1)⁸:

EE (%) = [(W_i − W_s) / W_i] × 100 (1)

where W_i is the initial amount of FUCO, and W_s is the amount detected in the supernatant⁸.

2.5. In vitro release profile:

The release behaviour of FUCO from nanoparticles was evaluated in simulated gastrointestinal (GI) fluids using a dialysis method as described previously¹⁰. Simulated gastric fluid (SGF, pH 1.2), simulated intestinal fluid (SIF, pH 6.8), and simulated colonic fluid (SCF, pH 7.4) were prepared with 30% (v/v) ethanol to enhance FUCO solubility and minimize aggregation¹¹. A 20 mL nanoparticle dispersion was loaded into dialysis bags (MWCO 14 kDa) and immersed in 100 mL of dissolution medium⁹. Free FUCO served as the control. The samples were sequentially incubated in SGF (0–2 h), SIF (2–5 h), and SCF (5–8 h) at 37 °C with agitation at 150 rpm. At predetermined intervals, 2 mL of medium was withdrawn and replaced with fresh medium to maintain sink conditions. FUCO content was quantified at 450 nm using a UV–Vis spectrophotometer. DDSolver software¹² was used to fit the release data to various kinetic models, and the best-fitting model was determined based on adjusted R², MSC, and AIC values.

2.6. Stability studies:

The storage stability of FUCO-COANPs was evaluated at 4 °C and 25 °C as described previously¹³. Samples were collected on days 5, 15, 30, 45, 60, and 90 and analyzed for PS and EE using the same procedures described in Section 2.4.

2.7. In vitro bioaccessibility:

The bioaccessibility of FUCO from both free and nanoparticle-loaded forms was evaluated using a static in vitro digestion model adapted from Shah et al.¹⁴. Simulated salivary, gastric, and intestinal fluids were prepared according to standard compositions. During the oral phase, 20 mL of the sample was mixed with simulated salivary fluid (SSF) and incubated at 37 °C for 10 min¹⁴. This was followed by gastric digestion with simulated gastric fluid (SGF, pH 2.5) for 2 h and intestinal digestion (pH 7.0) in the presence of bile and calcium for another 2 h. After digestion, the samples were centrifuged, and the aqueous micellar phase was collected. FUCO concentration in this phase was measured at 450 nm to calculate bioaccessibility using Equation (2)¹⁴:

Bioaccessibility (%) = (C_Micelle/C_Initial) x 100 (2)

where C_Micelle is the FUCO concentration in the micellar phase and C_Initial is the initial FUCO¹⁴.

2.8. In vitro cytotoxicity Assay:

The cytotoxicity of FUCO-COANPs, free FUCO, and blank COANPs was evaluated in Caco-2 colorectal cancer cells (ATCC, USA) using the MTT assay, following the method of Muangnoi et al.¹⁵. Cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin at 37 °C in a 5% CO₂ atmosphere. A seeding density of 3 × 10⁴ cells/well was used in 96-well plates. Blank COANPs were tested at concentrations of 5-100 % (v/v) to determine non-toxic levels. After 24 h of exposure, the medium was replaced, and cells were incubated with 0.5 mg/mL MTT solution for 4 h. The resulting formazan crystals were dissolved in DMSO, and absorbance was measured at 540 nm. Cell viability was calculated using Equation (3)¹⁵:

Cell viability (%) = (OD_sample/OD_control) ×100 (3)

The highest concentration of blank COANPs showing no significant cytotoxicity (p > 0.05) compared to untreated controls was selected for further evaluation. FUCO-COANPs and free FUCO were then administered at equivalent FUCO concentrations ranging from 5 to 30 μg/mL. After 24h of treatment, cell viability was reassessed using the MTT assay to compare the cytotoxic potential of encapsulated and free FUCO.

2.9. In vitro antioxidant activity:

The radical-scavenging activity of FUCO and FUCO-COANPs was determined using the DPPH assay according to Navarro-Hoyos et al.¹⁶ with slight modifications. Samples (10-150 μg/mL) were mixed with 0.25 mM ethanolic DPPH and incubated in the dark for 30 min at 25 °C. Absorbance was measured at 517 nm, and scavenging activity was calculated using Equation (4):

(A_control − A_sample)

DPPH scavenging activity (%) = –––––––––––––– ×100 (4)

A_control

where A_control is the absorbance of the DPPH solution without the sample, while A_sample refers to the absorbance observed in the presence of the test sample.

2.10. In vitro anti-diabetic activity:

α-Amylase inhibition was measured using a colorimetric assay modified from Telagari and Hullatti¹⁷. Acarbose or FUCO formulations (10-150 µg/mL) were separately combined with buffer and enzyme, incubated for 10 min, followed by addition of starch substrate and further reaction for 1 h. DNS reagent was added to terminate the reaction, and the mixture was boiled for 5 min and diluted. Absorbance at 540 nm was recorded and inhibition was calculated using Equation (5):

Inhibition (%) = [(A_control − A_sample)/A_control] × 100 (5)

where A_control refers to the absorbance of the reaction without inhibitor, and A_sample denotes the absorbance in the presence of the tested formulation.

2.11. Statistical Analysis:

All data are presented as the mean ± SD from triplicate experiments. Statistical significance was evaluated by one-way ANOVA followed by Scheffé’s post hoc test using SPSS software (IBM Corp., USA). A p-value of < 0.05 was considered statistically significant.

3. RESULTS AND DISCUSSION:

3.1. Statistical analysis:

The Box–Behnken design (BBD) generated a wide range of nanoparticle characteristics under different formulation conditions. PS varied from 258 ± 25 nm to 479 ± 34 nm, while EE ranged from 63.8 ± 1.9% to 87.2 ± 1.1%, as shown in Table 2. Multiple regression analysis was applied to obtain predictive equations describing the effects of independent variables on PS and EE. The predictive equations are presented in Equations (6) and (7):

PS (Y₁) =

357 + 46.6X₁ + 4.88X₂ - 0.25X₃– 1.23X₁X₂ + 9X₁X₃+ 52.5X₂X₃ - 2.38X₁² – 45.37X₂² + 66.38X₃² …..(6)

EE (Y₂) = 74 + 7.38X₁ – 5.5X₂ + 1.62X₃ ……..(7)

As shown in Figure 1, the 3D response surface plots (3D-RSM) revealed that the CSO:ALG mass ratio (X₁) had a significant effect on both PS and EE (p < 0.05). Increasing X₁ levels tended to enlarge PS, likely due to the formation of thicker polymeric layers around ALG, consistent with previous findings⁹. Although Tween™ 80 (X₂) slightly increased PS, the change was not statistically significant (p > 0.05) and may be associated with improved emulsification stability rather than droplet coalescence¹⁰. Interestingly, higher FUCO concentrations (X₃) reduced PS, possibly because hydrophobic interactions among FUCO molecule compact nanoparticle formation, as also reported for other hydrophobic compounds such as capsaicin, nerolidol, and lovastatin⁷. EE was positively influenced by both X₁ and X₃ (p < 0.05), while X₂ exhibited a negative effect, likely due to viscosity interference during nanoparticle self-assembly²⁵. The statistical adequacy of the models was verified by ANOVA (Table 3), where both R² and adjusted R²values exceeded 0.80, confirming the robustness and predictive reliability of the Box–Behnken optimization.

3.2. Formulation optimization and model validation:

To determine the optimal formulation that minimizes particle size (PS) and maximizes encapsulation efficiency (EE), numerical optimization was performed using Design-Expert^® software. The desirability function approach was employed to identify the most suitable combination of formulation parameters, as summarized in Table 4. The optimized conditions included a CSO:ALG mass ratio of 0.12:1, 2.5% (w/v) Tween™ 80, and FUCO concentration of 5 mg/mL. Under these conditions, the software predicted a PS of 346 nm and an EE of 86.3%. Model validation was carried out experimentally, yielding observed values of 339 ± 23 nm for PS and 89.2 ± 1.1% for EE, which showed no statistically significant differences from the predicted outcomes (p > 0.05). The close agreement between predicted and experimental results supports the reliability of the optimization strategy for developing FUCO-loaded nanocarriers with desirable physicochemical characteristics.

3.3. Characterization of FUCO-COANPs:

The optimized FUCO-COANPs exhibited a PS of 339 ± 23 nm and an EE of 89.2 ± 1.1%, closely matching the predicted values (346 nm and 86.3%) from the statistical design (Table 4). The measured polydispersity index (PDI) of 0.41± 0.03 indicated an acceptable particle size distribution for the optimized formulation²⁷. The zeta potential (ZP) was +32.5 ± 0.8 mV, suggesting strong electrostatic repulsion that contributes to colloidal stability²⁸. This value exceeds the ± 20 mV benchmark typically associated with preventing aggregation in aqueous dispersions²⁸. Transmission electron microscope (TEM; Figure 2a) revealed spherical nanoparticles with uniform surface morphology. The particle size observed under TEM appeared slightly smaller than that measured by DLS, likely due to dehydration and shrinkage of the hydrated layers during TEM vacuum preparation.²⁸^,²⁹ Collectively, these findings support the successful formation of FUCO-COANPs with physicochemical characteristics suitable for their potential use as oral drug delivery systems.

3.4. In vitro release study:

The release behavior of FUCO-COANPs and free FUCO was evaluated under simulated gastrointestinal (GI) conditions to assess formulation performance (Figure 2b). In the gastric phase (0–2 h), free FUCO released approximately 50%, whereas FUCO-COANPs released only 27%, indicating that the nanoparticle matrix limited early FUCO release under acidic conditions³⁰. In the intestinal phase (2–5 h), FUCO-COANPs exhibited sustained release, reaching 54% at 5 h, while free FUCO rapidly exceeded 83%, highlighting the benefit of nanoparticle-mediated sustained release, particularly in the intestinal region, an essential site for lipophilic drug absorption⁴⁷. During the colonic phase (5–8 h), FUCO-COANPs demonstrated controlled release behavior, achieving 85% cumulative release. These results suggest that FUCO-COANPs are suitable for phase-dependent sustained drug release under simulated GI conditions. To describe the release kinetics, experimental data were fitted to various mathematical models using DDSolver software. The Korsmeyer–Peppas model provided the best fit, as indicated by the highest adjusted R² and Model Selection Criterion (MSC) values and the lowest Akaike Information Criterion (AIC)³¹. The release exponent (n = 0.596) indicated a non-Fickian diffusion mechanism governed by both diffusion and polymer matrix relaxation³², which is characteristic of polymer-based nanoparticle systems.

Figure 2: (a) TEM micrograph of FUCO-COANPs fabricated under optimized parameters; (b) in vitro drug release profiles of both free FUCO and FUCO-COANPs evaluated in simulated GI media; (c) in vitro assessment of bioaccessibility for free FUCO and encapsulated FUCO after simulated digestion; (d) evaluation of FUCO-COANPs storage stability at 4 °C and 25 °C, over a 90-day period (n = 3). Values represented by identical letters do not differ significantly (p > 0.05).

Table 5: Kinetic modeling on FUCO was released from FUCO-COANPs.

Model	Evaluation criteria
Model	R²*_adjusted*	AIC	MSC	*k_n*	n	m
Zero-order (F = k₀.t)	0.855	57.576	1.156	8.725	-	-
First-order (F = 100. e-^k1.t)	0.955	47.044	2.327	0.129	-	-
Korsmeyer-Peppas (F = k_KP.tⁿ)	0.967	45.128	2.541	17.920	0.596	-

3.5. In vitro bioaccessibility:

Comparative bioaccessibility (BA) analysis was performed for free and encapsulated FUCO under simulated gastrointestinal conditions (Figure 2c). Free FUCO showed limited BA (22.4 ± 2.8%), consistent with its low aqueous solubility and instability in physiological environments³³^,³⁴. In contrast, FUCO-COANPs significantly enhanced FUCO bioaccessibility (61.8 ± 3.4%, p < 0.05), nearly tripling that of the free form. This improvement may be attributed to the nanoparticle structure, which may help maintain FUCO in a dispersed state and facilitate incorporation of FUCO into mixed micelles during digestion³². Additionally, the CSO–ALG complex may have contributed to improved solubilization through mucoadhesive and electrostatic interactions³⁶^,³⁷. These findings demonstrate the potential of COANPs to enhance the gastrointestinal bioaccessibility of poorly soluble compounds such as FUCO.

3.6. Stability study:

The storage stability of FUCO-COANPs was monitored for 90 days at 4 °C and 25 °C (Figure 2d). Nanoparticles stored under refrigerated conditions maintained consistent particle size and EE throughout the study period. In contrast, significant changes were observed from day 30 onward at 25 °C (p < 0.05), suggesting thermal sensitivity. These alterations may result from particle aggregation or matrix softening induced by temperature-related relaxation, which could disrupt nanoparticle integrity and promote drug leakage³⁸^,³⁹. These results emphasize the importance of cold storage to preserve the physicochemical stability of FUCO-COANPs in during formulation storage.

3.7. In vitro cytotoxicity:

The cytotoxicity of FUCO-COANPs, free FUCO, and blank COANPs was evaluated in Caco-2 cells using the MTT assay. Given the dose-dependent nature of nanoparticle toxicity⁴⁰, preliminary screening was first performed to identify concentrations compatible with cell viability. As shown in Figure 3a, blank COANPs at concentrations up to 20% (v/v) did not significantly affect cell viability compared with untreated controls (p > 0.05), indicating their cytocompatibility under the tested conditions. Subsequently, cells were treated with FUCO and FUCO-COANPs at equivalent FUCO concentrations (5-30 µg/mL). Figure 3b demonstrates a concentration-dependent decrease in cell viability for both formulations. Control groups treated with 0.5% DMSO (99.7 ± 2.3%) and blank COANPs (99.8 ± 1.9%) showed negligible cytotoxicity, indicating minimal contribution from the blank formulation. Notably, FUCO-COANPs exhibited significantly greater cytotoxic activity than free FUCO at all tested doses (p < 0.05), reducing cell viability to 38.6 ± 3.7% at 30 µg/mL compared with 54.9 ± 4.2% for free FUCO. This enhancement is likely attributed to improved FUCO dispersion, formulation–cell interaction, and protection of FUCO from degradation ⁴¹^,⁴². These findings suggest that FUCO-COANPs potentiate FUCO’s cytotoxic activity against colorectal cancer cells.

Figure 3: (a) cell viability of Caco-2 cells following treatment with blank COANPs; (b) comparative MTT assay results for free FUCO and FUCO-COANPs; (c) DPPH radical scavenging activity of free FUCO and FUCO-COANPs; (d) α-amylase inhibitory activity of free FUCO, FUCO-COANPs, and acarbose. Data are presented as mean ± SD (n = 3). Different letters indicate statistically significant differences (p < 0.05).

3.8. In vitro antioxidant activity:

The antioxidant potential of FUCO-COANPs and free FUCO was evaluated using the DPPH assay. Blank COANPs exhibited negligible scavenging activity (<3%), indicating minimal contribution from the blank nanocarrier. Both FUCO formulations demonstrated significant, dose-dependent free radical scavenging effects (p < 0.05), as shown in Figure 3c. FUCO-COANPs achieved a scavenging capacity of 62.8 ± 1.5% at 150 µg/mL, representing a 2.3-fold enhancement over free FUCO (27.6 ± 1.8%). This improvement can be attributed to enhanced solubility, nanoscale dispersion, and increased molecular accessibility of FUCO within the polymeric matrix⁴³^,⁴⁴, enabling more efficient interactions with DPPH radicals. These results support the potential of FUCO-COANPs to enhance the in vitro antioxidant performance of FUCO.

3.9. In vitro anti-diabetic activity:

The α-amylase inhibitory activity of FUCO-COANPs was assessed in comparison with free FUCO and acarbose across concentrations of 10–150 µg/mL. All samples exhibited a concentration-dependent inhibition pattern. FUCO-COANPs demonstrated significantly greater α-amylase inhibition than free FUCO at all tested concentrations (p < 0.05). At 150 µg/mL, FUCO-COANPs achieved 88.3 ± 2.8% inhibition, notably higher than free FUCO (65.2 ± 3.1%) and comparable to acarbose (96.5 ± 2.2%, p > 0.05). A similar trend was observed at 100 µg/mL, with FUCO-COANPs yielding 69.6 ± 1.7% inhibition compared with 55.1 ± 1.4% for free FUCO and 77.1 ± 2.5% for acarbose. Blank COANPs exhibited negligible α-amylase inhibition (< 5%), indicating that the inhibitory activity was mainly attributable to FUCO. The superior inhibitory effect of FUCO-COANPs is likely due to improved solubilization and stabilization of FUCO, which may enhance enzyme accessibility and interaction⁴⁵^,⁴⁶. These findings support the applicability of FUCO-COANPs as a nanocarrier-based approach for enhancing the in vitro α-amylase inhibitory activity of FUCO.

4. CONCLUSION

This study developed FUCO-COANPs, optimized via design-based formulation strategies. The optimized nanocarriers exhibited favorable physicochemical characteristics, acceptable particle distribution, and improved in vitro biological performance. These characteristics included enhanced antiproliferative activity in Caco-2 cells, increased antioxidant and α-amylase inhibitory activities, and improved bioaccessibility and release behavior of FUCO under simulated gastrointestinal conditions. Collectively, these findings indicate that FUCO-COANPs may serve as a promising oral delivery system for improving the functional performance of lipophilic compounds such as FUCO. The formulation holds potential for further development in applications related to oxidative stress, metabolic dysfunction, and colorectal cancer models. Further pharmacokinetic and toxicological evaluations will be necessary to support future in vivo and translational development.

5. AUTHOR CONTRIBUTIONS

Thanyaphon Pothi: Investigation, data curation, formal analysis, and writing – original draft. Chote Werawong: Resources, materials preparation, and investigation support. Niyom Laoopugsin: Investigation support and sample preparation. Chatchana Phroedphring: Technical assistance, nanoparticle characterization support, and data acquisition. Narissara Kulpreechanan: Conceptualization, methodology, validation, interpretation of results, and writing – review and editing. Chaturawit Boonpitak: Data organization, visualization support, and manuscript verification. Feuangthit N. Sorasitthiyanukarn: Conceptualization (lead), methodology (lead), supervision (lead), project administration, validation, writing – review and editing, and correspondence. All authors read and approved the final manuscript.

6. CONFLICT OF INTEREST:

The authors declare that they have no known commercial, financial, or personal conflicts of interest that could have influenced the design, execution, or reporting of this study.

7. ACKNOWLEDGMENTS:

The authors gratefully acknowledge the Office of Research and Academic Services, Western University, Thailand, for its financial support through a research grant. This support contributed to the completion of this work.

8. AI-ASSISTED WRITING DECLARATION:

Generative AI and AI-assisted tools were used only for language refinement and grammar checking. The authors reviewed and approved the final manuscript and take full responsibility for its cont

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Received on 08.07.2025 Revised on 12.11.2025

Accepted on 14.01.2026 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):3199-3206.

DOI: 10.52711/0974-360X.2026.00455

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Response	p-value	R²	*R²_adjusted*	*R²_predicted*	Lack of fit	Adeq Precision	Remark
Y₁
Linear model	0.2203	0.3191	0.1338	-0.4478	0.0287		-
2FI model	0.3832	0.5257	0.1699	-1.4852	0.0275		-
Quadratic model	< 0.0001	0.9941	0.9835	0.9663	0.8731	33.4658	Suggested
X₁exerted statistically significant effects (p < 0.05) on the Y₁
Y₂
Linear model	< 0.0001	0.9057	0.8800	0.8047	0.2582	19.3986	Suggested
2FI model	0.3714	0.9349	0.8861	0.6812	0.2538		-
Quadratic model	0.9783	0.9372	0.8242	0.0784	0.1410		-
X₁, and X₃exerted statistically significant effects (p < 0.05) on the Y₂

Factor	Composition	Response	Predicted	Observed	%Error
CSO:ALG	0.12:1	PS (nm)	346	339 ± 23	- 2.1
Tween™ 80	2.5 %w/v	EE (%)	86.3	89.2 ± 1.1	+ 3.3
FUCO	5 (mg/mL)