Enhancement of the Interaction between Bovine Serum Albumin and Quercetin by Quaternary Ammonium Chitosan: Multispectroscopic Insights into Photophysics, Bioaccessibility, and Bioactivity

Abstract

Quercetin (Que)-encapsulated bovine serum albumin (BSA) nanoparticles incorporated with quaternary ammonium chitosan (HACC) were prepared successfully. The nanoparticles were characterized by Fourier transform infrared (FT-IR) spectroscopy, and their morphology was examined using scanning electron microscopy (SEM). Dynamic light scattering (DLS) analysis showed that the Que-BSA-HACC nanoparticles ranged in size from 233 to 276 nm with an encapsulation efficiency of up to 62%. The effects of HACC on the interaction between BSA and Que were investigated by using multispectroscopic techniques. The introduction of HACC increased the binding constant of the complex, indicating an enhanced binding affinity. The Que-BSA-HACC interaction was predominantly driven by hydrophobic and electrostatic interactions. Moreover, the nanoparticles exhibited an enhanced quercetin release under simulated gastrointestinal conditions. The nanoparticles also exhibited enhanced antioxidant and anti-inflammatory activities compared to free Que, which was attributed to improved aqueous dispersibility. In contrast, cytotoxicity studies against SW-620 cells revealed that blank BSA-HACC nanoparticles exhibited comparable antiproliferative effects, suggesting that the carrier matrix plays a major role in the observed activity. These findings contribute to the development of novel flavonoid-based delivery systems.

1. Introduction

Quercetin (Que) is a polyphenolic flavonoid that is abundant in fruits, vegetables, and medicinal herbs. In recent years, it has attracted considerable attention because of its broad spectrum of biological activities, including antioxidant, anti-inflammatory, anticancer, cardioprotective, and neuroprotective effects. , Despite its high therapeutic potential, the application of Que is limited by several unfavorable physicochemical properties, most notably its poor water solubility, low oral bioavailability, rapid metabolism, and chemical instability under physiological conditions. , Nanotechnology-based delivery systems have emerged as an effective strategy for enhancing the therapeutic performance of bioactive compounds with poor solubility. Nanoparticles offer several advantages, such as an increased surface area, enhanced solubility, improved stability, a prolonged circulation time, and potential for targeted delivery. Encapsulation of Que within nanoparticles can protect it from environmental degradation, improve its systemic absorption, and enable controlled or site-specific release, thereby enhancing its overall bioefficacy. − Various nanocarriers have been explored for Que delivery, including polymeric nanoparticles, lipid-based systems (e.g., liposomes and solid lipid nanoparticles), micelles, and inorganic nanomaterials. −

Among these, there has been increased interest in protein–polysaccharide hybrid nanoparticles due to their excellent biocompatibility, biodegradability, and structural versatility. Bovine serum albumin (BSA) is a well-established carrier protein widely used in nanoparticle fabrication and is widely employed as a model protein because of its high structural and sequence homology with human serum albumin (HAS), sharing approximately 76% sequence identity and ligand binding domains. Therefore, the interaction trends observed in the present study are expected to provide useful insights for HAS-base delivery systems. It can efficiently encapsulate hydrophobic molecules through hydrophobic interactions, exhibit pH-responsive behavior, and its surface can be easily modified. The interactions between drugs and albumins play a crucial role in drug distribution, stability, and pharmacological efficacy, as they are closely related to the molecular structure and binding affinity. Therefore, understanding drug–albumin interactions is essential to elucidate drug behavior, pharmacokinetics, and structure–function relationships. , Environmental factors such as temperature, pH, and ionic strength can induce conformational changes in proteins, potentially leading to structural destabilization and reduced complex stability. , The incorporation of polysaccharides with proteins can significantly enhance the functional properties of the resulting complexes, enabling their application in drug delivery systems, biomaterial substitutes, and food packaging. , Polysaccharide–protein interactions have been shown to improve protein properties such as rheological behavior, dispersibility, and emulsification capacity.

Natural biopolymers, such as chitosan and cellulose derivatives, have attracted considerable attention because of their biocompatibility, biodegradability, and versatile functional properties. Quaternary ammonium chitosan, commonly referred to as 2-hydroxypropyltrimethylammonium chloride chitosan (HACC), is a water-soluble chitosan derivative, widely used in nanoparticle formations. Compared to native chitosan, HACC exhibits improved water solubility and enhanced antibacterial activity. The electrostatic interactions between negatively charged BSA and positively charged HACC promote the formation of stable compact nanoparticles. Furthermore, the HACC cation enhances mucoadhesion, cellular uptake, and biological activity, making it particularly suitable for drug delivery applications. ,

Similarly, cellulose and its derivatives, such as carboxymethyl cellulose, have been extensively utilized as reinforcing agents in active packaging materials due to their excellent mechanical properties and ability to form stable matrices. Recent studies have shown that lignin-cellulose-based composites can improve film functionality, offering promising applications in sustainable food packaging systems. In addition, biopolymer-based encapsulation systems have gained increasing interest for the delivery of bioactive compounds. Encapsulation using polysaccharide-protein matrices has been reported to enhance stability, protect against harsh gastrointestinal conditions, and improve the physicochemical properties of functional formulations. These findings underscore the potential of combining protein and polysaccharide components, such as BSA and modified chitosan, to develop effective delivery systems for hydrophobic bioactive compounds.

Fluorescence-based photophysical techniques provide powerful and sensitive tools for investigating drug–protein interactions at the molecular level. Albumin–drug interactions are commonly studied by using fluorescence quenching analysis, which is particularly effective for elucidating binding mechanisms. BSA exhibits intrinsic fluorescence due to its tryptophan (Trp) and tyrosine (Tyr) residues, serving as sensitive probes for monitoring microenvironmental changes upon ligand binding. In addition, Que exhibits enhanced fluorescence when bound to albumin, and this phenomenon can offer valuable insights into binding modes, quenching mechanisms, and conformational changes within protein-based nanoparticle systems. ,

Although BSA- and chitosan-based quercetin delivery systems have been reported, the influence of HACC on the molecular interaction between BSA and quercetin and its contribution to nanoparticle formation and performance remain poorly understood. Therefore, the present study aimed not only to develop BSA-HACC nanoparticles for quercetin delivery but also to elucidate the role of HACC in modulating the interaction between BSA and quercetin by using spectroscopic techniques. The molecular interaction was subsequently correlated with the physicochemical properties, encapsulation efficiency, simulated gastrointestinal behavior, antioxidant activity, anti-inflammatory activity, and anticancer activity of the resulting nanoparticles. These findings provide new insights into the design of protein–polysaccharide hybrid nanocarriers for drug delivery.

2. Experimental Section

2.1. Materials

Quercetin dehydrate (>96.0%), pepsin, and pancreatin were purchased from TCI (Tokyo, Japan). BSA was obtained from Thermo Fisher Scientific. Sodium tripolyphosphate (TPP), sodium phosphate monobasic dehydrate, sodium hydrogen phosphate, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich. HACC with a degree of substitution of >98% and a degree of acetylation of >90% was purchased from Chanjao Longevity Co. Ltd. (Bangkok, Thailand). All solvents were of analytical grade, purchased from Labscan (Bangkok, Thailand), and used directly without further purification.

2.2. Preparation of Que-BSA-HACC Nanoparticles

A concentrated BSA stock solution (300 mg/mL) was prepared by dissolving BSA in 0.01 M phosphate-buffered saline (PBS, pH 7.4); it was stirred (700 rpm) at room temperature for 2 h. Appropriate volumes of the stock solution were then diluted to obtain the desired BSA concentrations for nanoparticle preparation. Que was dissolved in ethanol and slowly added dropwise to the BSA solution over 15 min, followed by stirring at 700 rpm for 2 h to allow complete ethanol evaporation. HACC (20 mg/mL) and TPP (0.4 mg/mL) were mixed in a 1:4 (v/v) ratio. Then, the appropriate volume of HACC solution was slowly added dropwise into the BSA-Que solution over 15 min and stirred (700 rpm) at room temperature for 1 h. The mixture was centrifuged to remove unencapsulated Que. Que-loaded BSA-HACC nanoparticles that were obtained were used for subsequent experiments. Each formulation was repeated three times to check reproducibility.

2.3. Characterization

A dynamic light scattering laser particle analyzer (Malvern, UK) was used to measure the size and zeta potential of the nanoparticles. Attenuated total reflection–Fourier transform infrared (ATR-FTIR) spectrometry was performed with a Spectrum II device (PerkinElmer) to analyze functional groups and intermolecular interactions. The surface morphology of the nanoparticles was examined with scanning electron microscopy (SEM), using a Quanta 450 device.

2.4. Ultraviolet–Visible (UV–vis) Spectroscopy

A Lambda 365 UV–vis spectrophotometer (PerkinElmer) was used to examine absorption at 200–800 nm.

2.5. Fluorescence Spectroscopy

Fluorescence emission spectra were measured using a fluorescence spectrophotometer (FL8500, PerkinElmer).

2.5.1. Intrinsic Fluorescence Spectroscopy

The Stern–Volmer equation was used to assess fluorescence quenching changes: ,

$$\frac{F_0}{F}=1+k_{\mathrm{q}}{\tau }_0[Q]=1+K_{\mathrm{S}\mathrm{V}}[Q]$$ 1

where F and F ₀ are the fluorescence intensities with and without Que; k _q is the quenching rate constant; τ ₀ is the fluorescence lifetime of the fluorophore without the quencher; [Q] is the concentration of the quencher; and K _SV is the Stern–Volmer quenching constant.

The fluorescence intensities were corrected for the inner-filter effect before calculating the Stern–Volmer quenching constant (K _sv) and binding parameters. The corrected fluorescence intensity was obtained using the following equation:

$$F_{\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{r}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{e}\mathrm{d}}=F_{\mathrm{o}\mathrm{b}\mathrm{s}\mathrm{e}\mathrm{r}\mathrm{v}\mathrm{e}\mathrm{d}}\times {10}^{(A_{\mathrm{e}\mathrm{x}}-A_{\mathrm{e}\mathrm{m}})/2}$$ 2

where F _corrected and F _observed are the corrected and experimentally observed fluorescence intensity, respectively, and A _ex and A _em are the absorbance values at the excitation and emission wavelengths, respectively.

The binding constant (K _a) and the number of binding sites (n) were calculated using logarithmic equations.

$$\log \left(\frac{F_0-F}{F}\right)=\log K_{\mathrm{a}}+n\log [Q]$$ 3

Thermodynamic parameters were calculated from the Van’t Hoff equation to determine the main interactions between Que and BSA or HACC:

$$\ln K_{\mathrm{a}}=-\frac{\Delta H}{\mathit{RT}}+\frac{\Delta S}{R}$$ 4

$$\Delta G=\Delta H-T\Delta S$$ 5

when ΔS, ΔH, and ΔG are the entropy change, enthalpy change, and Gibbs free energy, respectively; R is the gas constant (R = 8.314 J/mol/K); and T is the thermodynamic temperature.

2.5.2. Synchronous Fluorescence Spectroscopy

Synchronous fluorescence spectra of BSA in the absence and presence of Que, and the BSA-Que complex in the absence and presence of HACC, were measured at 298 K. The intervals between the excitation and emission wavelengths (Δλ) were fixed at 15 and 60 nm, corresponding to the microenvironment around Tyr and Trp residues, respectively. The emission wavelength scan range was recorded from 230 to 350 nm.

2.6. Encapsulation Efficiency (EE)

The EE of Que in the nanoparticles was determined using UV–vis spectroscopy. The remaining Que was removed after centrifuge. The concentration of Que encapsulated in the nanoparticles was calculated from the UV absorbance using a Que calibration curve. All experiments were done in triplicate. The EE was calculated using the following equation:

$$\mathrm{EE}(\%)=\frac{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{a}\mathrm{p}\mathrm{s}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{Q}\mathrm{u}\mathrm{e}\mathrm{i}\mathrm{n}\mathrm{N}\mathrm{P}\mathrm{s}}{\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{Q}\mathrm{u}\mathrm{e}\mathrm{u}\mathrm{s}\mathrm{e}\mathrm{d}\mathrm{f}\mathrm{o}\mathrm{r}\mathrm{N}\mathrm{P}\mathrm{s}\mathrm{p}\mathrm{r}\mathrm{e}\mathrm{p}\mathrm{a}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}}\times 100$$ 6

2.7. Release in Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF)

SGF and SIF were prepared on the basis of a published study. The SGF electrolyte stock solution contained 0.5 M KCl (6.9 mL), 0.5 M KH₂PO₄ (0.9 mL), 1 M NaHCO₃ (12.5 mL), 2 M NaCl (11.8 mL), 0.15 M MgCl₂(H₂O)₆ (0.4 mL), and 0.5 M (NH₄)₂CO₃ (0.5 mL), with the pH adjusted to 3 by using 6 M HCl. Then, the volume was adjusted to 500 mL. Five milliliters of SGF was prepared by mixing 3.8 mL of the SGF electrolyte stock solution, 0.2 mL of pepsin (2 000 U/mL), 0.3 mL of CaCl₂, and 0.7 mL of water. The SIF electrolyte stock solution was prepared by combining 0.5 M KCl (6.8 L), 0.5 M KH₂PO₄ (0.8 mL), 1 M NaHCO₃ (42.5 mL), 2 M NaCl (9.6 mL), and 0.15 M MgCl₂(H₂O)₆ (1.1 mL) with the pH adjusted to 7 by using 6 M HCl. The volume of the SIF electrolyte stock solution was adjusted to 500 mL. Five milliliters of SIF was prepared similar to SGF, except that pancreatin (100 U/mL) replaced pepsin.

The sample was placed into a dialysis membrane (cutoff 12 kDa) and immersed in 5 mL of SGF or SIF at a temperature of 37 °C. Subsequently, at scheduled intervals, 200 μL of solution was withdrawn and replaced with the same volume of solution. The amount of Que released was detected by UV–vis spectrometry at 330 nm and calculated from a Que calibration curve. All measurements were done in triplicate.

2.8. Antioxidant Activity

DPPH radical scavenging was determined based on the method described by Xiao et al. A DPPH stock solution (8.0 mg in 100 mL absolute ethanol) was prepared and stored in the dark. For the assay, 100 μL of sample was added to the wells of a 96-well microplate, followed by 100 μL of the DPPH stock solution. The plate was incubated at room temperature in the dark for 30 min. The absorbance of the sample (A _sample) and control (A _control) at 517 nm was recorded using a Spark 10 M microplate reader. PBS was used as a control. All samples were run in triplicate. The radical scavenging efficiency of samples was calculated using eq ():

$$\mathrm{DPPH}\mathrm{scavenging}\mathrm{efficiency}=\frac{A_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}-A_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}}{A_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}\times 100$$ 7

2.9. Anti-Inflammatory Activity

Cell viability was determined based on the WST-1 assay, as described previously, to estimate the probable cytotoxic effect of the test compound and positive controls (0.1% sodium dodecyl sulfate [SDS]) on human keratinocyte cells (HaCaT, CLS-300493, CLS, Germany). The number of viable cells was determined based on the ability of mitochondria to convert the tetrazolium salt WST-1 to water-soluble formazan. The amount of water-soluble formazan generated by dehydrogenase in a cell is directly proportional to the number of viable cells present and can be measured. Briefly, HaCaT cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin–streptomycin in a humidified incubator with 5% CO₂ at 37 °C. When the cells reached 80% confluency, they were trypsinized for 5 min, counted with a hemocytometer, seeded into the wells of a 96-well plate at 2 × 10⁵ cells per mL (100 μL per well), and then incubated overnight. The cells were then treated with various concentrations of the samples for 24 h. After incubation, the medium was removed and replaced with 100 μL of WST-1 solution (Abcam, USA), and the plate was incubated at 37 °C for 30 min. Then, the absorbance was measured at 450 nm with a microplate reader (Infinite M200 PRO, TECAN, USA). All experiments were performed in triplicate. Cell viability was calculated using eq ():

$$\mathrm{c}\mathrm{ell}\mathrm{viability}(\%)=\frac{{\mathrm{A}\mathrm{b}\mathrm{s}}_{\mathrm{t}}}{{\mathrm{A}\mathrm{b}\mathrm{s}}_{\mathrm{c}}}\times 100$$ 8

where Abs_t and Abs_c represent the absorbance of treated cells and untreated cells, respectively.

To examine anti-inflammatory activity, HaCaT cells were seeded at 5 × 10⁵ cells per mL in 24-well plates. Then, they were incubated for 24 h with samples and 100 mg/mL of D,L-10 acetoxychavicol acetate (DL-1) or β-glucan as a positive control. After incubation, PBS was used to wash the cells and replace the culture medium. The cells were exposed to UVA (314–400 nm, 3.0 W) and UVB (280–315 nm, 13.6 W) radiation for 22 s. Then, PBS was replaced with culture medium, and the cells were incubated for another 24 h. After incubation, inflammatory cytokine levels in the cell culture supernatants were quantified using an enzyme-linked immunosorbent assay (ELISA). Absorbance was read at 450 nm with a microplate reader. All standards and samples were assayed in triplicate.

2.10. Anticancer Activity

The metastatic human colorectal cancer cell line SW-620 was cultured in Roswell Park Memorial Institute 1640 medium (RPMI-1640; Gibco, Thermo Fisher Scientific, USA), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Gibco, Thermo Fisher Scientific, USA) at 37 °C in a humidified 5% CO₂ incubator (Binder, Germany). The SW-620 cell line was kindly provided by Dr. Mattaka Khongkaw, Senior Researcher at the National Nanotechnology Center (NANOTEC), Thailand.

The cytotoxic effects of BSA-HACC, QueNPs, free Que, and DMSO were evaluated using the MTT assay. SW-620 cells were seeded in 96-well plates at a density of 5 × 10⁴ cells/mL and incubated for 24 h. MTT solution (5 mg/mL) was then added, followed by incubation at 37 °C for 2 h. After removal of the supernatant, formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm using a microplate reader (BioTek Instruments, USA). Cell viability was expressed as a percentage relative to untreated controls, and IC₅₀ values were determined.

2.11. Statistical Analysis

All experiments were performed using at least three independent preparations of samples. For physicochemical characterization, including particle size, polydispersity index (PDI), zeta potential, encapsulation efficiency, release studies, and antioxidant activity assays, measurements were conducted in triplicate and the results are presented as mean ± standard deviation (SD).

For cell-based assays, including cytotoxicity and anti-inflammatory activity, three independent biological experiments were performed, with each treatment evaluated in triplicate wells. The results are expressed as mean ± SD.

Statistical analyses were carried out using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc multiple comparison test. Differences were considered statistically significant at P < 0.05.

3. Results and Discussion

Que was encapsulated by binding to BSA to form the Que-BSA complex. Then, HACC was added to stabilize the Que-BSA nanoparticles through ionic interactions between the negatively charged BSA and the positively charged HACC. Because the isoelectric point (pI) of BSA is approximately 4.5–4.8, BSA carries a net negative charge at pH 7.4; therefore, nanoparticle formation was performed in PBS at pH 7.4 (Scheme ).

1. Schematic Diagram of Que-BSA-HACC Nanoparticle Formation.

3.1. Effect of BSA on the Absorption and Emission of Que

UV–vis absorption and fluorescence spectroscopy were used to analyze the photophysical behavior of the Que-BSA complex. Increasing concentrations of BSA were added to the Que solution in PBS (pH 7.4). There was a gradual decrease in the UV absorption band at 370 nm, accompanied by the appearance and increase of a new absorption band at 425 nm (Figure a). These spectral changes provide clear evidence of Que-BSA complex formation. Furthermore, the presence of an isosbestic point at 399 nm indicates the coexistence of two spectrally distinct species in solutionfree Que and BSA-bound Quesuggesting the formation of a single, well-defined complex.

1.

(a) UV absorption spectra and (b) fluorescence emission spectra of 33 μM Que in PBS (pH 7.4) in the presence of BSA (0–120 μM).

The fluorescence emission spectra of Que are shown in Figure b. A major emission band at 531 nm corresponds to the excited state intramolecular proton transfer (ESIPT) tautomers of Que, while a weaker emission band at 427 nm is attributed to the neutral form of Que. In the presence of BSA, the fluorescence emission intensity increased progressively as the BSA concentration increased and exhibited a red shift to 537 nm, which can be attributed to stabilization of the tautomeric form of Que during Que-BSA complex formation. Que, which contains 5-OH and 3-OH groups adjacent to the carbonyl group (CO), is inherently weakly fluorescent in aqueous media. The intramolecular hydrogen bond between the 5-OH group and CO interferes with ESIPT from C(3)–OH···OC(4), resulting in low fluorescence emission. However, biomacromolecules such as proteins, DNA, and RNA disturb the intramolecular hydrogen bond between the 5-OH group and CO, thereby facilitating ESIPT from C(3)–OH···OC(4). This phenomenon enhances fluorescence emission upon binding to a target or within a more rigid microenvironment. , Overall, the observed enhancement of tautomer fluorescence in the presence of BSA indicates a strong binding interaction between Que and BSA.

3.2. Effect of HACC on the Absorption and Emission of Que

Figure a shows the UV–vis absorption spectra of Que upon the addition of increasing concentrations of HACC in the absence of BSA. The characteristic absorption band of Que at 370 nm gradually decreased and shifted slightly to 375 nm, while a new absorption band at 450 nm emerged and increased in intensity. The spectral changes suggest the formation of a Que-HACC complex. Figure c presents the UV–vis absorption spectra of Que in the presence of BSA with the subsequent addition of varying concentrations of HACC. In this case, the absorption band at 450 nm increased slightly in intensity, indicating the formation of a Que-BSA-HACC ternary complex.

2.

(a) UV–vis absorption and (b) fluorescence emission spectra of 6 μM Que with various concentrations of HACC (in PBS, pH 7.4) and without BSA. (c) UV–vis absorption and (d) fluorescence emission spectra of 6 μM Que with various concentrations of HACC (in PBS, pH 7.4) in the presence of 25 μM BSA. The insets show Stern–Volmer plots of the fluorescence quenching of (b) 6 μM Que and (d) Que-BSA by HACC.

Figure b,d shows the fluorescence emission spectra of Que in the presence of different concentrations of HACC and in the absence and presence of BSA, respectively. The quenching of Que was further analyzed using Stern–Volmer plots. The fluorescence intensity of Que gradually decreased as the HACC concentration increased, regardless of whether BSA was present, indicating that HACC interacts with Que. In the absence of BSA, Que bound directly to HACC primarily through hydrogen bonds, resulting in a higher Stern–Volmer quenching constant (K _sv = 1.33 ×10⁵ M^–1). However, K _sv was lower in the presence of BSA (K _sv = 0.46 × 10⁵ M^–1). This result suggests that most Que molecules initially bind to BSA to form the Que-BSA complex. Upon the subsequent addition of HACC, the interaction between Que and BSA is further stabilized, and the Que-BSA complex associates with HACC through ionic interaction between negatively charged BSA and positively charged HACC. Consequently, the quenching efficiency of HACC toward Que is reduced in the presence of BSA, leading to a lower K _sv.

3.3. Analysis of Binding and Thermodynamic Parameters

The spatial structure of proteins is usually reflected in their fluorescence parameters, including the emission intensity, emission wavelength, and quantum yield, particularly those associated with Tyr and Trp residues. Fluorescence spectroscopy was used to examine the structural changes induced by interactions between BSA, Que, and HACC. The addition of HACC caused no significant change in the fluorescence intensity of BSA, but there was a slight red shift in the maximum emission wavelength (Figure a–c). This shift indicates that the presence of HACC increased the hydrophilicity of the microenvironment surrounding the Tyr and Trp residues in BSA. These effects became more pronounced as temperature increased from 298 to 338 K.

3.

Fluorescence spectra of the Que-BSA complex with HACC (0, 0.01, and 0.1%, w/v) at (a) 298 K, (b) 318 K, and (c) 338 K. (d–f) Stern–Volmer plots and (g–i) the logarithmic equation fitting curves.

The binding affinity between BSA and Que is a key factor in understanding the interaction. It can be assessed with parameters including K _a and n. Proteins exhibit intrinsic fluorescence due to their aromatic amino acids, and this intrinsic fluorescence is highly sensitive to changes in the polarity of the surrounding microenvironment. Fluorescence quenching can occur when proteins interact with small molecules or quenchers. To gain insights into the structural changes induced by interactions among BSA, Que, and HACC, the fluorescence emission spectra of the BSA-Que complex were investigated at varying HACC concentrations and temperatures.

Fluorescence quenching mechanisms between proteins and Que are normally classified as dynamic and static. Dynamic quenching involves collisional interactions in which the excited fluorophore returns to its ground state, resulting in decreased fluorescence intensity. In contrast, static quenching arises from the formation of a non-fluorescence ground-state complex between the protein and fluorophore, leading to reduced fluorescence emission. In dynamic quenching, an increase in temperature typically enhances molecular collisions, leading to an increase in the quenching constant (k _q) and the slope of the Stern–Volmer plot. Conversely, static quenching is characterized by a decrease in the quenching constant with an increase in temperature due to the dissociation of the ground-state complex. All fluorescence emission spectra are shown in the supporting information, Figure S1, and the quenching mechanism was evaluated using Stern–Volmer plots. The slopes of these plots decreased significantly for all samples as temperature increased, indicating that the fluorescence quenching of BSA by Que predominantly occurs via a static quenching mechanism (Figure d–f). Moreover, the presence of HACC did not alter the quenching mechanism between BSA and Que, suggesting that HACC does not disrupt the formation of the BSA-Que ground-state complex.

K _a and n were calculated using eq () and are presented in Table . K _a is widely used as an indicator of the binding affinity between proteins and ligands, with higher K _a values corresponding to stronger binding interactions. K _a greater than 10⁴ M^–1 indicates a strong binding affinity and favorable complex formation between hydrophobic molecules and proteins. The results showed that the K _a followed the order of BSA-Que < BSA-Que-HACC 0.01% < BSA-Que-HACC 0.1% at all investigated temperatures, indicating that the presence of HACC significantly enhanced the binding affinity between BSA and Que. Temperature also had an effect on K _a and n. K _a of the ternary complex increased as temperature increased, suggesting that the interaction between Que and BSA is an endothermic process and the stability of the complex improves at higher temperatures. Based on the relatively large K _a, the complexation of Que with BSA in the presence of high concentrations of HACC is highly favorable. This behavior may be attributed to the disordered and flexible native structure of BSA. Notably, the K _a values obtained in this study are comparable to those previously reported for Que interacting with soy protein isolate in the presence of sodium alginate (3.02–6.17 × 10⁵ L/mol) and show a similar increase (284.12–418.12 × 10⁵ L/mol) when the temperature was raised to 338 K.

1. Stern–Volmer Quenching Constant (K _sv), Quenching Constant (K _q), Binding Constant (K _a), and Thermodynamic Parameter of BSA-Que and BSA-Que-HACC Complexes at Different Temperatures.

sample	T/K	K sv × 104 M–1	K q × 1010 M–1s–1	K a × 104 M–1	n	ΔH kJ M–1	ΔS kJ M–1	ΔG kJ M–1
BSA-Que	298	1.24	2.09	5.598	1.60	28.09	0.127	–9.62
	318	0.82	1.38	7.244	1.18			–12.15
	338	0.71	1.20	21.84	1.87			–14.68
BSA-Que-HACC 0.01%	298	1.51	2.54	21.902	1.16	15.21	0.094	–12.86
	318	1.05	2.06	17.277	1.28			–14.75
	338	0.88	1.51	46.512	1.70			–16.63
BSA-Que-HACC 0.1%	298	1.70	2.85	33.020	1.29	20.57	0.118	–14.59
	318	0.95	3.47	72.72	2.13			–16.95
	338	0.83	1.39	87.196	2.17			–19.31

The value of n was approximately 1, indicating that BSA binds to Que in a 1:1 ratio. The apparent binding site number (n) increased with temperature and HACC concentration, suggesting more complex binding behavior between Que and BSA in the presence of HACC. However, because n is derived from an empirical fluorescence binding model, it cannot be interpreted as direct evidence for the presence of two discrete binding sites. Under these conditions, the molecular flexibility of BSA increases, promoting the exposure of specific amino acid residues that facilitate additional Que-binding sites.

Thermodynamic parameters derived from steady-state fluorescence measurements at various temperatures were also investigated to elucidate changes in the noncovalent interactions between BSA and Que. A negative ΔG indicates that the formation of the ternary complex is a spontaneous process. A positive ΔH values suggest that the binding interactions of the ternary complex are endothermic, which explains the observed increase in K _a as the temperature increased. Moreover, the positive ΔH and ΔS values are generally considered consistent with a contribution from hydrophobic interactions to the binding process. However, because these thermodynamic parameters were derived indirectly from fluorescence-based binding data, they should be interpreted cautiously, as hydrophobic interactions are the dominant driving force.

It should be noted that the Van’ Hoff analysis applied in this study assumes a temperature-independent enthalpy change (ΔH) over the investigated temperature range (298–338 K). While this approximation is commonly used in fluorescence-based studied of protein–ligand interactions over relatively narrow temperature intervals, it may not fully account for temperature-induced conformational changes in proteins such as BSA. Therefore, the obtained thermodynamic parameters should be considered as apparent values that provide qualitative insight into the dominant interaction forces rather than absolute thermodynamic quantities. ,,

3.4. Synchronous Fluorescence and Circular Dichroism Spectroscopy (CD)

Synchronous fluorescence spectroscopy was used to investigate microenvironmental changes around Tyr and Trp residues in BSA induced by interactions with Que and HACC. Spectra were recorded at a Δλ of 15 and 60 nm to determine polarity changes around Tyr and Trp residues, respectively. As the Que concentration increased, λ_max of Trp residues (Δλ = 60 nm) exhibited a red shift, indicating an increase in the polarity of the microenvironment surrounding Trp residues (Figure a). This implies a more hydrophilic microenvironment around Trp residues due to Que binding. In contrast, there was not a significant λ_max shift for Tyr residues (Δλ = 15 nm; Figure b). Figure c,d presents the synchronous fluorescence spectra of the BSA-Que complex in the presence of HACC. Upon the addition of HACC, there was a slight blue shift in λ_max of Trp residues (Δλ = 60 nm), and a red shift for Tyr residues (Δλ = 15 nm). These spectral changes indicate alterations in the polarity of the microenvironment around Trp and Tyr residues of BSA resulting from interactions with HACC. Notably, there was no shift in the synchronous fluorescence spectra for BSA when the HACC concentration increased (Supporting Information, Figure S2). These findings demonstrate that HACC interacts with BSA but does not significantly alter the microenvironment of aromatic residues of BSA. This implies that HACC does not directly interact with the primary hydrophobic binding sites of BSA.

4.

Synchronous fluorescence spectra of (a and b) 1 mg/mL BSA with varying concentrations of Que and (c and d) the 1 mg/mL BSA 0.01 mg/mL Que complex with varying concentrations of HACC, (e) CD spectra, (f) secondary structure content of BSA, BSA-HACC, BSA-Que, and BSA-Que-HACC nanoparticles and (g) a schematic diagram of the binding behavior of Que bound to BSA and interaction with HACC during the formation of nanoparticles.

Circular dichroism (CD) spectroscopy provided further insight into the conformational changes of BSA upon interaction with HACC and Que. CD spectra and secondary structure content of BSA, BSA-Que, BSA-HACC, and BSA-Que-HACC nanoparticles are shown in Figure e,f, respectively. The addition of HACC led to a decrease in α-helix content and an increase in the random coil content, indicating partial unfolding of the protein, which can be attributed to strong electrostatic interactions between the cationic HACC and negatively charged BSA. In contrast, the presence of Que in both BSA-Que and BSA-Que-HACC nanoparticles increased α-helix and random coil content, suggesting a stabilization effect on the protein structure. In addition, the summary of the proposed interaction of Que, BSA, and HACC during nanoparticle formation is shown in Figure g.

Although the combination of UV–vis absorption, steady-state fluorescence, synchronous fluorescence, circular dichroism (CD), and thermodynamic analyses consistently supports the proposed interaction mechanism between quercetin, BSA, and HACC, indicating that HACC modifies the conformation of BSA and enhances its interaction with quercetin. Previous studies have shown that quercetin preferentially binds to Site I (subdomain IIA) of native BSA using competitive binding assays with warfarin as a Site I marker. In the present study, our primary objective was to elucidate the influence of HACC on the interaction between BSA and quercetin during nanoparticle formation rather than to identify the binding site. Since HACC induces conformational changes in BSA, as evidenced by the CD results, the binding characteristics of quercetin in the BSA-HACC system may differ from those of native BSA. Nevertheless, the spectroscopic techniques employed provide indirect evidence of the binding process and cannot definitively resolve the molecular details of the interactions. Therefore, complementary approaches, including site-marker competitive binding assays, fluorescence lifetime measurements, isothermal titration calorimetry (ITC), molecular docking, or molecular dynamics simulations, would provide complementary information regarding the binding mechanism and interaction energetics. These investigations were beyond the scope of the present study but will be considered in future work to further validate the proposed mechanism on the molecular level.

3.5. Nanoparticle Characterization and Encapsulation Efficiency (EE)

Quantitative particle size analysis was performed by measuring 50 nanoparticles from the SEM image using ImageJ software. Figure a shows an SEM image of Que-BSA-HACC nanoparticles, which clearly exhibited spherical morphology with an average diameter of 219 ± 21.80 nm, which is consistent with DLS results. Furthermore, the interactions between BSA, Que, and HACC were investigated using FT-IR spectroscopy (Figure b). The BSA spectrum showed characteristic absorption bands at 1637 and 1527 cm^–1 corresponding to the amide I band (CO stretching) and the amide II band (C–N stretching coupled with N–H bending), respectively. , The characteristic peaks of HACC appeared at 3291 and 1643 cm^–1, which are attributed to O–H stretching and CO stretching of the N-acetyl amide bond, respectively. Que exhibited CO stretching at 1664 cm^–1 and CC stretching of the aromatic ring at 1610 cm^–1. After nanoparticle formation, the C–H stretching of the CH₂ and CH₃ groups in BSA shifted from 2953 to 2963 cm^–1. Additionally, the amide I band of BSA shifted from approximately 1637 to 1652 cm^–1, while the amide II band shifted from 1527 to 1534 cm^–1. These spectral shifts confirmed the structure modification of BSA upon complexation, driven by electrostatic and hydrophobic forces established in the thermodynamic analysis.

5.

(a) SEM image of Que-BSA-HACC nanoparticles and (b) FT-IR spectra of BSA, HACC, Que, Que-BSA-HACC nanoparticles (QueNPs), and a physical mixture of BSA-HACC-Que.

The size of Que-BSA-HACC nanoparticles was analyzed using DLS (Figure a–c). The particle size decreased as the Que concentration increased up to 80 μg/mL, likely due to saturation of Que loading. Increasing the BSA concentration did not significantly affect the nanoparticle size, which was optimized at approximately 245 nm at a BSA concentration of 4 mg/mL. Additionally, the HACC concentration had no substantial influence on particle size. However, the PDI values ranged from 0.4 to 0.8, indicating a relatively broad particle size distribution and considerable heterogeneity of the particle population. This behavior is likely associated with the complex nature of the BSA-HACC nanoparticle system due to multiple intermolecular interactions, which is an important limitation of the present formulation. This suggests that further optimization of the formulation and preparation conditions is required to obtain a more uniform nanoparticle system. Furthermore, particle aggregation of the nanoparticles, supported by SEM observations and the relatively low zeta potential values, may also contribute to the increased PDI values.

6.

Size and polydispersity index (PDI) of nanoparticles with varying concentrations of (a) Que, (b) BSA, and (c) HACC. The zeta potential of nanoparticles with varying concentrations of (d) Que, (e) BSA, and (f) HACC. EE of Que in nanoparticle with varying concentrations of (g) Que (BSA 0.6 mg/mL, HACC 20 μg/mL), (h) BSA (Que 20 μg/mL, HACC 20 μg/mL), and (i) HACC (Que 20 μg/mL, BSA 2.0 mg/mL). Markers of different letters in the figure denote that the mean difference is significant at p < 0.05.

The ζ-potential values are presented in Figure d–f. In the absence of HACC, the nanoparticles exhibited a negative ζ-potential, which is consistent with the presence of deprotonated carboxyl groups on the protein surface at neutral pH. Upon incorporation of HACC, a cationic polysaccharide, the ζ-potential shifted toward less negative values, indicating partial charge neutralization. This shift confirms the electrostatic interaction between the negatively charged BSA and positively charged HACC and suggests the formation of a stable polyelectrolyte complex. However, the ζ-potential values range from +9 to −10 mV, indicating that the nanoparticles possess a near-neutral surface charge. This suggests that the electrostatic stabilization is limited in this system. We further clarified that the slight negative charge likely arises from residual deprotonated groups of BSA, while the overall nearly neutral values indicate charge compensation between negatively charged BSA and cationic HACC, confirming the formation of a polyelectrolyte complex. Despite this, the hydrophilic polymeric backbone of HACC and the flexible loops of BSA project outward into the aqueous phase, binding a thick layer of water molecules. This highly hydrated polymeric shell generates physical steric hindrance that successfully counteracts attractive van der Waals forces. The colloidal stability is therefore attributed mainly to steric stabilization provided by the biopolymer matrix rather than electrostatic repulsion. However, this interpretation is based on indirect observations, and additional studies are required to directly confirm the contribution of the steric stabilization mechanisms. Moreover, the relatively high PDI values may be attributed to the nature of the protein–polysaccharide nanoparticle system in which multiple interactions can lead to the formation of particles with varying sizes. In addition, partial aggregation of nanoparticles, as suggested by SEM observations and low ζ-potential values, may also contribute to the increased PDI.

The EE of Que encapsulated in BSA-HACC nanoparticles was investigated using UV–vis spectroscopy (Figure g–i). Increasing the Que concentration decreased the EE, likely due to the limited binding capacity of BSA. In contrast, the EE of Que increased from approximately 10 to 60% as the BSA concentration increased, indicating enhanced Que–protein interactions. Variation in the HACC concentration had no significant effect on the EE, which only reached a maximum of approximately 62% at the highest concentration. These results suggest that Que primarily binds to BSA during nanoparticle formation, followed by the subsequent incorporation of HACC onto the BSA-Que nanoparticles. This interpretation is consistent with Stern–Volmer quenching results described above, in which K _sv of Que in the presence of both BSA and HACC was lower than that of direct Que-HACC interaction, indicating stronger binding of Que to BSA. Increasing the HACC concentration produced only a slight increase in binding affinity, while particle size and encapsulation efficiency showed no significant improvement. Therefore, 20 ug/mL HACC was selected for further experiments as the optimized formulation because it provided sufficient nanoparticle formation while minimizing unnecessary polymer content.

The stability of the nanoparticles was evaluated by monitoring the particle size over time at room temperature. The average particle size increased from 238 to 587 nm over 18 days, indicating gradual aggregation during storage (data shown in Supporting Information, Figure S3). These findings suggest that the nanoparticle system exhibits limited long-term stability under test conditions, which is consistent with the relatively low ζ-potential values observed, indicating weak electrostatic stabilization. Although the nanoparticle dispersions remained stable without immediate macroscopic precipitation, further formulation optimization is required to improve storage stability for practical application. Possible stabilization strategies may include storage under refrigerated conditions or conversion into dried, re-dispersible formulations. Despite this, the nanoparticle formulation remains suitable for short-term use or fresh preparation, for example, in functional food or beverage systems.

3.6. Release in SGF and SIF

A dialysis bag method was used to evaluate the sustained release behavior of Que-BSA-HACC nanoparticles in SGF and SIF compared with free Que (Figure a). The results demonstrate an enhanced quercetin release from the nanoparticles under SGF and SIF conditions. Moreover, the physicochemical properties of QueNPs were further evaluated under simulated gastrointestinal conditions (data shown in Supporting Information, Figure S4). The average particle size of QueNPs increased to 325 nm after incubation in SGF and further increased to 382 nm in SIF, while the PDI and zeta potential remained nearly unchanged. The increase in particle size is likely due to swelling of the protein–polysaccharide matrix and partial particle aggregation during simulated gastrointestinal digestion. These results suggest that the nanoparticles retained their overall structural integrity under gastrointestinal conditions while undergoing moderate swelling, which may facilitate the sustained release of the encapsulated Que. The enhanced release observed under SGF and SIF conditions suggests the potential of the nanoparticles to improve the availability of quercetin during gastrointestinal transit; however, the direct bioaccessibility was not evaluated in the present study.

7.

(a) Release profiles of Que-BSA-HACC and Que from dialysis bags in SGF and SIF. (b) DPPH radical scavenging activity of Que and Que-BSA-HACC nanoparticles (QueNPs). Markers of different letters in the figure denote that the mean difference is significant at p < 0.05.

3.7. Biological Activities

3.7.1. DPPH Radical Scavenging Activity

The antioxidant activity of Que loaded in BSA-HACC nanoparticles at various concentrations (0.5–4 μg/mL) was evaluated based on the DPPH radical scavenging assay in PBS (pH 7.4). Que-BSA-HACC nanoparticles exhibited higher apparent radical scavenging activity than free Que under the same experimental conditions (Figure b). However, it should be noted that Que has limited solubility in aqueous media such as PBS. In contrast, encapsulation within the BSA-HACC nanoparticle system significantly improves the aqueous dispersibility of Que, resulting in enhanced apparent antioxidant performance. The higher apparent antioxidant activity of QueNPs compared to free Que may be attributed to improve aqueous dispersibility of quercetin in the nanoparticles. Therefore, the observed difference in antioxidant activity is particularly improved solubility and dispersion of Que, rather than an intrinsic increase in its radical scavenging ability. These findings highlight the role of the nanoparticle system in enhancing the practical functionality of hydrophobic bioactive compounds in aqueous environments.

3.7.2. Anti-Inflammatory Activity

The WST-1 assay was used to examine the viability of HaCaT cells in the presence of free Que and Que-BSA-HACC nanoparticles (Figure a). The concentration used in the anti-inflammatory assay was selected based on the cytotoxicity results, which demonstrated greater than 80% cell viability. Therefore, a concentration of 0.15 μg/mL was chosen for anti-inflammatory studies. Based on the encapsulation efficiency of 47.50 ± 1.17% of the QueNP formulation containing 2.0 mg/mL BSA and 20 μg/mL HACC, the amount of QueNP formulation required to deliver 0.15 μg/mL Que was calculated. Free Que was then tested at the same Que concentration (0.15 μg/mL) to enable a direct comparison of anti-inflammatory activity.

8.

(a) Percent cell viability in the presence of Que and Que-BSA-HACC nanoparticles (QueNPs). The levels of the inflammatory molecules (b) TNF-α, (c) IL-6, (d) PGE2, and (e) IL-10 for the Que, QueNPs, and BSA-HACC nanoparticles without Que (NP blank). NP blank was prepared at the same concentration as QueNPs. Markers of different letters in the figure denote that the mean difference is significant at p < 0.05.

UVB exposure induces inflammation in keratinocytes, leading to the activation of several signaling pathways. Activated keratinocytes produce proinflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin 1 (IL-1), IL-6, IL-8, and interferon γ (IFN-γ), along with other inflammatory mediators including nitric oxide (NO) and prostaglandin E₂ (PGE2), which contribute to cellular inflammatory responses. In this work, after treatment with free Que, Que-BSA-HACC nanoparticles, or BSA-HACC nanoparticles without Que (NP blank), HaCaT cells were exposed to UVA/UVB radiation. The resulting cytokine and mediator levels are presented in Figure . DL-1 and β-glucan were used as reference standards.

Compared with the non-UV-treated control, UV-exposed cells showed significantly higher levels of inflammatory cytokines (Figure c–e). These results demonstrate that both free Que and Que-BSA-HACC nanoparticles effectively reduce the production of proinflammatory cytokines and mediators and enhance IL-10 secretion, which play a crucial role in suppressing inflammatory responses. NP blank did not significantly reduce TNF-α, IL-6, or PGE2 levels, nor did they enhance IL-10 production. This confirms that the observed anti-inflammatory activity is primarily attributed to the presence of Que in the nanoparticle formulation.

However, the improved antioxidant and anti-inflammatory activities are likely attributable to improved aqueous dispersibility, encapsulation, and gastrointestinal bioaccessibility of quercetin rather than solely to an increased binding constant. Although HACC enhanced the binding affinity between BSA and quercetin, this stronger interaction should be considered one contributing factor that improves nanoparticle formation and stability, rather than direct evidence of an enhanced biological performance.

3.7.3. Anticancer Activity

The preliminary anticancer activity of QueNPs was evaluated and compared with that of NP blank and free Que against metastatic colorectal cancer SW-620 cells using the MTT assay (data shown in Supporting Information, Figure S5). Table summarizes the IC₅₀ values obtained after 24 h of treatment. Both BSA-HACC nanoparticles (at the same volume and concentration as those used in QueNPs) and QueNPs exhibited comparable inhibitory effects on cell proliferation. In contrast, free Que showed a higher IC₅₀ value. DMSO, used as a positive control, demonstrated a pronounced cytotoxicity. Notably, the blank BSA-HACC nanoparticles exhibited cytotoxicity comparable to that of QueNPs, indicating that the carrier itself substantially contributed to the observed antiproliferative effect. Therefore, the greater cytotoxicity observed for QueNPs relative to that of free Que cannot be attributed specifically to encapsulated quercetin. This observation suggests that the antiproliferative activity is predominantly driven by the carrier matrix itself rather than being solely dependent on Que-loaded nanoparticles. This pronounced inherent cytotoxicity of the blank carrier can be attributed to the structural features of HACC and BSA. First, HACC is a cationic charge that is well-established to have a significantly negative charge in cancer cell membranes. The strong electrostatic interaction between the positively charged HACC on the nanoparticle surface and the negatively charged SW-620 cell membrane can destabilize the lipid bilayer, inducing membrane permeability, leakage, and subsequent cell death. Second, because of rapidly proliferating cancer cells overexpressing albumin receptors to satisfy their metabolic nutrient requirements, the BSA-based formulation likely acts as an efficient endocytic vehicle. This receptor-mediated endocytosis promotes a massive intracellular accumulation of the nanoparticles, delivering toxic levels of the HACC polymer directly to the cytoplasm.

2. IC₅₀ Values of BSA-HACC (Blank NP), QueNPs, Free Que, and DMSO on SW-620 Metastatic Colorectal Cancer Cells.

treatments	IC50 (μg/mL)
BSA-HACC (blank NP)	90.0 ± 9.4
QueNPs	94.0 ± 4.7
free Que	134.8 ± 23.7
DMSO for positive control (%)	3.92 ± 0.36
DMSO (%, same conc. use for free Que)	NA

Therefore, the contribution of Que to the overall cytotoxic effect in nanoparticle formation cannot be clearly distinguished under the present conditions. Further investigations, such as dose-normalized comparisons or mechanistic studies, are required to clarify the specific role of Que in the system.

Although the developed BSA-HACC nanoparticles showed promising physicochemical properties and biological activity, several challenges remain before their practical application. Future studies should focus on optimizing large-scale production, evaluating long-term storage stability, and investigating in vivo pharmacokinetics. Nevertheless, the favorable encapsulation efficiency, improved bioaccessibility, and enhanced biological performance observed in this study support the potential of this nanoparticle system for future biomedical applications.

4. Conclusion

In this work, a novel drug-delivery system based on nanoparticles containing BSA and HACC was successfully developed for Que encapsulation. The Que-BSA-HACC nanoparticles exhibited an average particle size of approximately 233 nm, and the EE increased as the BSA concentration increased, reaching a maximum of 62%. Spectroscopic and thermodynamic analyses suggested that HACC modulated the interaction between BSA and Que, with the observed thermodynamic parameters being consistent with a contribution from hydrophobic interactions and electrostatic interactions. However, these interaction mechanisms are based on indirect spectroscopic evidence and require further molecular-level validation, such as fluorescence lifetime spectroscopy, isothermal titration calorimetry, molecular docking, or molecular dynamics simulations, to identify the specific binding site of quercetin on BSA. Furthermore, the results demonstrated that the BSA-HACC delivery system provides strong antioxidant activity, enhances the anti-inflammatory efficacy of Que, and sustained release of Que under stimulated gastrointestinal systems. The anticancer study revealed that quercetin-loaded BSA-HACC nanoparticles exhibited antiproliferative activity against SW-620 cells; the comparable cytotoxicity observed for the blank BSA-HACC nanoparticles indicates that the carrier itself contributes substantially to the biological response. Therefore, the enhanced antiproliferative effect cannot be attributed solely to quercetin encapsulation. Further mechanistic investigations are required to clarify the respective roles of the carrier and the encapsulated quercetin and to fully evaluate the therapeutic potential of this nanoparticle platform. Although the present study demonstrates enhanced antioxidant and anti-inflammatory activities in vitro, the biological performance of the nanoparticles should be further validated in appropriate animal models. Future studies will investigate pharmacokinetics, biodistribution, therapeutic efficacy, and safety in vivo to establish the translational potential of the proposed delivery system. Overall, this study provides new insights into the interactions among flavonoid–protein–polysaccharide complexes and demonstrates the potential of BSA-HACC nanoparticles as a platform for improving the dispersibility and bioaccessibility of hydrophobic bioactive compounds. A limitation of the present formulation is the relatively high PDI and the gradual increase in particle size during storage, indicating heterogeneity and limited long-term colloidal stability that require further formulation optimization and evaluate practical application in pharmaceutical and food-related systems.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c02923.

• Fluorescence emission spectra of BSA at various temperatures and in the presence of different concentrations of Que and HACC; synchronous fluorescence spectra of BSA with varying concentrations of HACC; plot of particle sizes and PDI of QueNPs over 18 days; cell viability of SW-620 cells treated with different samples (PDF)

J.S.: Writing original draft, methodology, investigation, and data curation. W.T.: Writing original draft, investigation, methodology, and formal analysis. C.S.: Writing original draft, investigation, methodology, and formal analysis. V.W.: writing original draft, investigation, and data curation. T.L.: Investigation, formal analysis, and supervision. T.K.: Writingreview and editing, supervision, project administration, and funding acquisition.

The authors declare no competing financial interest.