Synthesis, structural characterization, and antitumor effect of quercetin-loaded chitosan nanoparticles-stabilized pickering emulsion via suppressing the PI3K/AKT/mTOR signaling pathway

1. Introduction

GLOBOCAN 2020 estimated a global cancer incidence of 19.3 million new cases and 10 million deaths in 2020, becoming one of the major factors of mortality worldwide (Sung et al., 2021). In many countries, the increasing incidence of cancer as a predominant cause of death partly reflects a significant decline in mortality from coronary heart disease and stroke relative to cancer (Bray et al., 2021). The rapid rise in both cancer incidence and mortality is largely attributed to population aging and growth, along with changes in the prevalence, frequency, and patterns of cancer-related risk factors, many of which are closely linked with socioeconomic development (Gersten et al., 2002).

Chemotherapy remains one of the most effective cancer treatments; however, cytotoxic agents indiscriminately target rapidly dividing cells, thereby damaging normal proliferative tissues such as gastrointestinal epithelium, hair follicles, and bone marrow (Ben-Arye et al., 2012). This results in the spectrum of adverse effects, including nausea, alopecia, stomatitis, peripheral neuropathy, cardiomyopathy, leukopenia, and even secondary malignancies (Dehelean et al., 2021; Guruvayoorappan et al., 2015; Hashemzaei et al., 2017). Combination chemotherapy has recently emerged as an optimal strategy in clinical trials due to its enhanced target selectivity, synergistic mechanisms, and reduced drug resistance (Wang et al., 2017). Nevertheless, such regimens are also associated with cumulative toxicities, drug–drug interactions, and increased financial and somatic burdens on patients (Mokhtari et al., 2017). These limitations highlight the urgent need to develop and explore alternative selective anticancer therapies.

Ethnopharmacology has long demonstrated significant potential in disease management (Vafadar et al., 2020), and numerous phytopharmaceutical compounds with potent anticancer properties have been identified (Wang et al., 2016). Epidemiological studies suggest that diets rich in fruits and vegetables may reduce cancer susceptibility. Among bioactive phytoconstituents, quercetin (QCT), a major plant-derived flavonoid, has shown promising anticancer activity across multiple cancer models. Due to its favorable safety profile and multitarget mechanisms, QCT is considered a potential therapeutic phytochemical for cancer treatment. However, its clinical application remains limited owing to poor bioavailability, low aqueous solubility, chemical instability, and limited intestinal permeability (Wang et al., 2016).

To improve the therapeutic efficacy of QCT, various formulation strategies have been developed. Encapsulating lipophilic quercetin in emulsion-based delivery systems has proven effective in enhancing its pharmacological activity. Nanoencapsulation, which involves entrapping a compound within wall materials at the nanometer scale, can remodel the physicochemical and biological properties of the encapsulated material by increasing surface area and altering particle characteristics (Aranaz et al., 2021; Kyriakides et al., 2021). Pickering emulsions represent an ideal colloidal system for fabricating and delivering anticancer drugs in nanoparticle form (Cardial et al., 2019).

Medium-chain triglycerides (MCTs) are valuable components for emulsion synthesis (Shah et al., 2016). Coconut oil contains more than 48% MCTs, with lauric acid (C12) comprising 45-53% of its total triglyceride content. Lauric acid is particularly advantageous for Pickering emulsion synthesis owing to its rapid metabolism and excellent biocompatibility (Chen et al., 2021). Following ingestion, lauric acid is predominantly transported to the liver, with minimal entry into the lymphatic system as chylomicrons, thereby reducing the risk of conditions such as atherosclerosis and cardiovascular disease. Its metabolism generates ketone bodies capable of crossing the blood-brain barrier to provide energy to muscle and brain tissues (Furuta et al., 2023).

Chitosan (CS), a natural biopolymer with inherent antimicrobial properties, has been extensively used as a promising drug-delivery vehicle. CS exhibits unique characteristics, including biodegradability and biocompatibility with human tissues, without causing allergic reactions or immune rejection. Chitosan nanoparticles (CSNPs) have demonstrated anticancer potential by enhancing immune function (Virmani et al., 2023). Owing to these versatile properties, CS has gained considerable attention in the pharmaceutical industry.

Accordingly, the present study aimed to fabricate and optimize CSNP-stabilized Pickering emulsions, characterize quercetin-encapsulated Pickering emulsions, and evaluate their anticancer activity as a drug-delivery system for quercetin.

2. Materials and Methods

2.2 Methods

2.2.1 Preparation of polymeric chitosan-tripolyphosphate (CSNPs) nanoparticles

CS–TPP nanoparticles were synthesized using the ionic gelation method (Shah et al., 2016). Briefly, a 3% (w/v) chitosan solution was prepared by dissolving chitosan (molecular weight >150 kDa) in 1% (v/v) acetic acid with constant magnetic stirring at 500 rpm and left overnight at room temperature. The pH of the chitosan solution was adjusted to 4.7-4.8. A 0.1% (w/v) TPP solution was then prepared and added dropwise to the chitosan solution to induce nanoparticle formation. The mixture was stirred continuously at 500 rpm for 120 min at room temperature, lyophilized using a freeze dryer, and stored at 4°C until use.

2.2.2 Formulation of quercetin-loaded pickering emulsions (CSNPs-QPE)

Nanoparticle-stabilized Pickering emulsions were prepared following the method of Shah et al. with minor modifications (Shah et al., 2016). The emulsions consisted of two phases: (1) an aqueous phase containing CSNPs and (2) an oil phase of medium-chain triglycerides (MCT). For the oil phase, 8 mg of quercetin was dispersed in 45 mL of MCT and stirred overnight to maximize dissolution. Undissolved quercetin was removed by centrifugation at 14,000 g for 10 min. The aqueous and oil phases (1:1 ratio) were combined in a glass vial and homogenized using a high-speed rotor-stator homogenizer at 10,000 rpm for approximately 3 min at room temperature (Fig. 1). The resulting emulsions were stored in glass bottles at room temperature for further analysis.

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Fig. 1.

Schematic representation of the structural organization of CSNPs-QPE.

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2.2.3 Characterization of quercetin-loaded Pickering emulsions

2.2.3.1 Ultraviolet-spectrophotometer

UV spectra of pure quercetin, CSNPs-QPE, and CSNPs-UPE were recorded in the range of 100–400 nm using a HITACHI U-2900 spectrophotometer (Rehman et al., 2023). Peak shifts were analyzed to confirm emulsion formation and quercetin encapsulation.

2.2.3.2 Microscopic imaging

The morphology of the emulsions was examined using a Cell Imager Invitrogen EVOS FL at 10X, 20X, and 40X magnifications.

2.2.3.3 Quasi-elastic light scattering

The polydispersity index (PDI), emulsion droplet size, and zeta potential of QPE were determined using a Nanotrac Wave-II Zetasizer at 25°C fixing a scattering angle of 90° (Chen et al., 2021). Distilled water served as the dispersing medium. Each sample was analyzed in triplicate, and mean particle sizes were reported.

2.2.3.4 Fourier transform infrared (FTIR) spectroscopy

Fourier Transform Infra-red (FTIR) analysis was done to confirm the synthesis of the emulsion (Chen et al., 2021). The samples were analyzed with an FTIR-8400 spectrophotometer within the wave number range of 400-4000 cm^-1.

2.2.3.5 Scanning electron microscopy (SEM)

Scanning electron microscopy was employed for the analysis of surface morphology and particle size of the samples. A small amount of each sample was placed on aluminum stubs, air-dried, coated with gold, and imaged (Chen et al., 2021).

2.2.4 Encapsulation efficiency (EE)

Encapsulation efficiency (indirect method) was determined as described by Jamil et al. (2016) with slight modifications (Jamil et al., 2016). Briefly, freshly prepared CSNPs-QPE samples were centrifuged at 10,000 rpm for 10 min, and the supernatant was carefully collected. The concentration of unencapsulated quercetin in the supernatant was measured spectrophotometrically at 374 nm using a calibrated HITACHI U-2900 UV–Vis spectrophotometer. A quercetin calibration curve (0.63-320 µg/mL) was prepared in the same solvent system and yielded R² = 0.9986. The encapsulation efficiency (%) was calculated as:

$\text{Percentage EE}=\frac{\text{ Quercetin added}-\text{Quercetin encapsulated}}{\text{Quercetin added }}\times 100$

where $Q_{\text{added}}$ is the total quercetin mass introduced into the formulation and $Q_{\text{free}}$ is the mass quantified in the supernatant. All measurements were performed in triplicate for each independent batch.

2.2.5 In vitro drug release assay

The release profile of quercetin was evaluated using the dialysis method (Shah et al., 2016). Emulsion samples (5 mL) were sealed in dialysis tubing and immersed in 45 mL phosphate-buffered saline (PBS) at pH 7.4 or 4.0 in 50 mL Falcon tubes. The samples were incubated at 37°C in a shaking incubator set at 120 rpm. At 30-min intervals, 3 mL aliquots of the release medium were withdrawn and replaced with fresh PBS. The drug release was monitored for up to 72 h.

2.2.6 Cell culture

Liver cancer cells (HepG2) were seeded at a density of 3×104 cells in Dulbecco’s Modified Eagle Media (DMEM). Fetal Bovine Serum (10%, FBS) was added to DMEM media as a supplement. Streptomycin (100 μg/mL) and penicillin (100 Units/mL) were added to the media. Cells were maintained in 5% CO₂ incubator at 37°C.

2.2.7 Cells passaging

The human liver cancer cell line (HepG2) was cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 1% antibiotics and 10% FBS. The cells were sub-cultured upon reaching approximately 80% confluency. The used media was aspirated, and 75 cm² culture flasks were washed with phosphate-buffered saline (1X PBS; 5 mL). Trypsin–EDTA (0.5 mL or 1 mL) was added to detach cells, followed by neutralization with fresh medium (3 mL). The cell suspension was centrifuged for 2 min at 3500 rpm. The resulting cell pellet was resuspended in 10 mL of culture medium in culture flasks of 75 cm² and incubated overnight with 5% CO₂ concentration at 37°C in a humidified incubator.

2.2.8 In vitro cytotoxicity (MTT assay)

Cell viability was assessed using the MTT assay with minor modifications (Bahuguna et al., 2017). HepG2 cells were exposed to varying concentrations of CSNPs-QPE, CSNPs-UPE, along with control medium, for 24 h. Subsequently, 10 µL of MTT solution (5 mg/mL in PBS) was added to each well and incubated to allow formation of formazan crystals. Absorbance was measured at 570 nm with a reference wavelength of 620 nm using a BioTek microplate reader. Cell viability (CV%) was calculated as:

$CV\%=\frac{Absorbance of sample at 570nm-Abs of sample at 620 nm}{Absorbance of control at 570nm-Abs of control at 620 nm}\times 100$

2.2.9 In vitro migration assay

Cell migration was evaluated using a scratch assay with minor modifications (Rehman et al., 2023). HepG2 cells (5 × 10⁵ cells/well) were seeded into 12-well plates and incubated for 24 h. A cross-shaped scratch was made using a micropipette tip, and wells were washed with PBS. Cells were then treated with CSNPs-UPE, CSNPs-QPE, or doxorubicin (positive control). Wound closure was imaged at 0 and 24 h using a high-resolution inverted microscope.

2.2.10 Genes expression analysis

The gene expression of the PI3K/Akt/mTOR signaling pathway was assessed to evaluate the effects of Pickering emulsions on HepG2 cell proliferation and angiogenesis (Chen et al., 2021). After 48 h of treatment with CSNPs-UPE, CSNPs-QPE, or doxorubicin, total RNA was extracted using TRIzol® (Life Technologies, Invitrogen). The RNA concentration and quality were measured using an ND-1000 spectrophotometer (Optiplex, USA). First-strand cDNA was synthesized from extracted RNA using M-MLV Reverse Transcriptase (200 U/L; Invitrogen). Quantitative real-time PCR (qRT-PCR) was performed on an ABI 7500 system using SYBR Green Master Mix and sequence-specific primers described previously (Rehman et al., 2025). GAPDH served as the internal housekeeping control. The relative gene expression was calculated using the comparative Ct (2⁻ΔΔCt) method, normalized to GAPDH expression. All reactions were performed in triplicate to ensure reproducibility.

3. Results

3.1 Synthesis of CSNPs-QPE

CSNPs-QPE were synthesized using the ionic gelation method at a chitosan-to-TPP ratio of 3:1 (Fig. 2). The formation of a white, milky suspension confirmed the interaction between the amine group (+NH₃) of chitosan and the phosphate group (PO₄⁻) of TPP.

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Fig. 2.

Schematic illustration of the synthesis process for quercetin-loaded and unloaded Pickering emulsions.

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3.2 Characterization of CSNPs-QPE

3.2.1 UV-Vis spectroscopy

UV–Visible absorption spectra were recorded to confirm emulsion formation and quercetin encapsulation using a HITACHI U-2900 spectrophotometer. Samples were scanned over the wavelength range of 100 to 400 nm. Pure quercetin exhibited a characteristic absorbance peak at 375 nm, whereas CSNPs-QPE showed distinct peaks at 378.8 nm and 341 nm (Fig. 3a). The observed red shift from 375 nm to 378.8 nm confirmed successful CSNPs-QPE formation. This special red shift is attributed to microenvironmental changes around the quercetin chromophore, likely resulting from hydrogen-bonding, polarity changes, and π–π stacking within the polymeric matrix. (Fig. 3a). The additional absorbance at ∼341 nm, along with the disappearance of free-quercetin signals in the supernatant, further supports that quercetin molecules were incorporated into the emulsion droplets rather than remaining freely dissolved in the oil phase. In contrast, the unloaded Pickering emulsions (CSNPs-UPE) exhibited a distinct absorption peak at 287 nm, corresponding to the presence of oil and aqueous phases without quercetin.

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Fig. 3.

(a) UV–Visible spectra of CSNPs-QPE, CSNPs-UPE, and pure quercetin. The observed peak shifts from 375 to 379 nm and from 330 to 341 nm indicate bonding interactions among the constituent compounds of CSNPs-QPE and CSNPs-UPE. (b) FTIR spectra of CSNPs-QPE and CSNPs-UPE. CSNPs-QPE: quercetin-loaded chitosan Pickering emulsion; CSNPs-UPE: unloaded chitosan Pickering emulsion.

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3.2.2 Fourier transform infra-red spectroscopy

FTIR analysis was performed to investigate chemical interactions and confirm emulsion synthesis. Moisture-free KBr pellets were used to avoid spectral noise. The FTIR spectra of CSNPs-UPE and CSNPs-QPE have been presented in Fig. 3(b).

• CS-TPP nanoparticles showed characteristic peaks at 3332 cm⁻¹ and 1640 cm⁻¹. Broad band attributed to O–H stretching (phenolic -OH of quercetin and surface hydroxyls) and N–H stretching from chitosan. A shift of the chitosan peak from 1630 cm⁻¹ to 1640 cm⁻¹ indicated successful nanoparticle formation. The observed upshift upon nanoparticle formation (1630 → 1640 cm⁻¹) is typical of electrostatic crosslinking with TPP (interaction of –NH₃⁺ with PO₄³⁻), supporting successful ionic gelation (Fig. 3b).

• Pure lauric acid exhibited peaks at 2853 cm⁻¹ and 2942 cm⁻¹ (–CH₃ and –CH₂ stretching), 931 cm⁻¹ (hydroxyl wagging), 723 cm⁻¹ (C–H bending), 1702 cm⁻¹ (carbonyl group), and 1292 cm⁻¹ (C–O stretching) (Jiang et al., 2018).

• Pure quercetin displayed unique peaks for stretching and aromatic bending between 1095–1614 cm⁻¹ and phenolic –OH bending between 1213–1429 cm⁻¹.

• CSNPs-UPE showed peaks at 2879, 2945, 916, 750, 1707, and 1286 cm⁻¹. 916, 750 cm^-1 peaks are associated with alkyl bending (lauric acid) and out-of-plane aromatic C–H wagging; their preservation in CSNPs-UPE indicates intact oil-phase signatures in the emulsion.

• CSNPs-QPE exhibited peaks at 3304, 1230, and 1448 cm⁻¹corresponding to phenolic –OH bending, confirming Pickering emulsion formation. The 1230 cm⁻¹ band in CSNPs-QPE specifically suggests quercetin’s phenolic C–O contribution or new C–O interactions at the particle interface. In CSNPs-QPE, the band at 3304 cm⁻¹ is consistent with phenolic OH involvement and often broadens/changes when hydrogen bonding occurs.

3.2.3 Dynamic light scattering (DLS)

The particle size and zeta potential of the formulations were determined using dynamic light scattering (DLS) (Fig. 4). The average particle sizes of CSNPs and CSNPs-QPE were 124 nm and 238 nm, respectively. The corresponding zeta potential values were +44.5 mV for CSNPs and +59.5 mV for CSNPs-QPE, indicating strong electrostatic repulsion and high colloidal stability. Both formulations (CSNPs and CSNPs-QPE) showed narrow particle size distribution with polydispersity indices of 0.1192 and 0.457, respectively (Table 1). The PDI value ≈0.46 for quercetin-loaded Pickering emulsions reflects a slight increase in size heterogeneity likely resulting from oil loading and emulsification processes.

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Fig. 4.

Measurement of size, PDI, and zeta potential of nano-formulations through DLS. (a) CSNPs nanoparticles, (b) CSNPs-QPE.

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Table 1. Characterization of CSNPs and CSNPs-QPE depicting size, zeta potential, PDI, %EE, and % in vitro release values.

Samples	Size (nm)	Zeta potential (mV)	PDI	%EE	% Drug Release
CSNPs	124	+ 44.5	0.12	-	-
CSNPs-QPE	238	+ 59.5	0.45	88.5±2.0 (n=3)	81 (pH 4.0) 67 (pH 7.4)

3.2.4 Optical imaging of CSNPs-QPE

The morphology of the emulsions was examined using a Cell Imager Invitrogen EVOS FL. As shown in Fig. 5, the emulsions appeared spherical and uniform, consistent with DLS results.

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Fig. 5.

Nano-formulations viewed on cell imager EVOS at 40X magnification (a) CSNPs-QPE (GFP filter). (b) CSNPs-QPE transmittance filter. (c) Cell morphology visualization using Scanning Electron Microscopy of CSNPs-QPE.

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3.2.5 Scanning electron microscopy

SEM analysis confirmed the size and surface morphology of CSNPs-QPE. The images revealed uniformly shaped spherical emulsions (Fig. 5), further supporting optical microscopy findings. Further, SEM images showed uniform, spherical particles with smooth surfaces and no obvious large-scale aggregation.

3.2.6 Encapsulation efficiency of CSNP-QPE

Encapsulation efficiency (EE) is a key parameter in nanodrug delivery. Quercetin was efficiently encapsulated within the oil phase of the emulsions, achieving an EE of 88.8% (Table 1). This high EE suggests that CSNPs-QPE can serve as an effective drug delivery system.

3.3 In vitro release profile of CSNPs-QPE

The release of quercetin from CSNPs-QPE was evaluated at two pH scales, i.e., pH 4.0 and pH 7.4 to simulate physiological and acidic tumor environments (Fig. 6). After 30 min, drug release was approximately 2% at pH 7.4 and 15% at pH 4.0, indicating initial diffusion resistance due to the emulsion bilayer. After 2.5 h, a marked increase in release, reaching 67% and 81% at pH 7.4 and 4.0, respectively, followed by a slower diffusion phase. Overall, a higher cumulative release at pH 4.0 indicated increased drug diffusion under acidic conditions and a sustained release pattern. Further, release kinetics analysis further revealed that at pH 4.0, the quercetin release from CSNPs-QPE showed a moderate correlation (R² = 0.9081) with the zero-order model, indicating a nearly constant release rate with slight deviations over time (Table 2). The first-order model exhibited an excellent fit (R² = 0.9594), demonstrating that the release rate is concentration-dependent. The Higuchi model showed a strong linear relationship (R² = 0.9471), confirming that Fickian diffusion through the polymeric matrix primarily governs release. The Korsmeyer–Peppas model depicted a strong linear correlation (R² = 0.9531) with a release exponent (n) of 0.20, indicating that the drug release mechanism follows Fickian diffusion, with the release rate mainly governed by diffusion through the matrix (Table 2).

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Fig. 6.

(a) Encapsulation Efficiency of CSNPs-QPE. Representing the R2 value, quercetin was encapsulated efficiently in the oil phase of the emulsion. The percentage encapsulation efficiency was calculated to be 88.8%. (b) Graphical representation of in vitro drug release profile for CSNPs-QPE at pH 7.4 and 4.0. A sustained drug release pattern was observed for CSNPs-QPE.

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Table 2. Kinetic modelling of quercetin release profile of encapsulated Pickering emulsions

Samples	Zero order R2	First order R2	Korsmeyer-Peppas R2	Higuchi R2
CSNPs-QPE (pH4.0)	0.9081	0.9594	0.9534 n=0.20	0.9471
CSNPs-QPE (pH7.4)	0.9428	0.9605	0.9611 n=1.12	0.9563

3.4 Anti-tumour activity

3.4.1 In vitro cytotoxicity in liver cancer cells

The cytotoxic effects of doxorubicin-loaded (CSNPs-QPE) and unloaded (CSNPs-UPE) emulsions were evaluated against HepG2 liver cancer cells using the MTT assay at concentrations ranging from 80 to 2.5 μg/mL. In 96 wells plates, cells with 80-85% confluency were treated with doxorubicin, CSNPs-UPE, and CSNPs-QPE. Cell viability results have been shown in Fig. 7. The IC₅₀ value of CSNPs-QPE was 20 μg/mL after 48 h of formulation exposure (Fig. 7b). At a concentration of 80 μg/mL, CSNPs-QPE inhibited more than 90% of the cell proliferation, comparable to doxorubicin (∼94%) (Fig. 7a). CSNPs-QPE exhibited significant dose-dependent inhibition of cancer cell growth, consistent with the sustained drug release profile.

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Fig. 7.

Percentage cell viability of HepG2 liver cancer cells treated with CSNPs-QPE, CSNPs-UPE, and Doxorubicin (Doxo). Cell viability was assessed following 24 h treatment with varying concentrations of each formulation. The results indicate that both CSNPs-QPE and CSNPs-UPE show reduced inhibitory effects at concentrations below 5 µg/mL, while a pronounced dose-dependent cytotoxicity was observed at higher concentrations. (a) A bar graph representing % cell proliferation of CSNPs-QPE, CSNPs-UPE and doxorubicin. (b) Cell viability profile depicting IC50 for CSNPs-QPE and doxorubicin (c) Cell viability profile depicting IC50 for CSNPs-UPE.

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3.4.2 Wound healing assay

Cell migration was assessed using a scratch assay at CSNPs-QPE concentrations of 5 μg/mL and 20 μg/mL. After 24 h, wound closure was 72%, 78%, and 100% for CSNPs-UPE, CSNPs-QPE, and doxorubicin, respectively, at 5 μg/mL (Fig. 8). At 20 μg/mL, wound closure was 69%, 74%, and 100% for CSNPs-UPE, CSNPs-QPE, and doxorubicin, respectively.

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Fig. 8.

Analysis of migration assay for CSNPs-UPE, CSNPs-QPE, and Doxo at 5 μg/mL and 20 μg/mL in HepG2 cells.

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3.4.3 Evaluating antitumor effect of CSNPs-QPE upon liver cancer cells through PI3K/Akt/mTOR pathway

The anticancer activity of CSNPs-QPE was further evaluated by analyzing the PI3K/Akt/mTOR signaling pathway in HepG2 cells. Cells were treated with CSNPs-UPE, CSNPs-QPE, or DOX for 48 h. DOX served as a positive control.

• PI3K Expression: CSNPs-QPE at 5 μg/mL significantly inhibited PI3K expression compared to the negative control and DOX at the same concentration (Fig. 9). CSNPs-QPE exhibited a lower IC₅₀ value (4.5 μg/mL) than DOX (6.67 μg/mL) for PI3K inhibition (Fig. 10).

• Akt Expression: Both DOX and CSNPs-QPE caused significant downregulation of Akt (p < 0.0001). At 5 μg/mL, CSNPs-QPE inhibited 81% of Akt expression, whereas DOX inhibited 75% (Fig. 11). The IC₅₀ values for Akt inhibition were 2.24 μg/mL for CSNPs-QPE and 3.43 μg/mL for DOX (Fig. 12).

• mTOR Expression: CSNPs-QPE induced marked downregulation of mTOR compared to CSNPs-UPE and DOX (Fig. 13). At 5 μg/mL, CSNPs-QPE inhibited >90% of mTOR expression (p < 0.0001), while DOX showed minimal inhibition at the same concentration. At 20 μg/mL, mTOR inhibition reached 90% with CSNPs-QPE and 75% with DOX. The IC₅₀ values were 2.24 μg/mL for CSNPs-QPE and 10.01 μg/mL for DOX (Fig. 14).

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Fig. 9.

Quantitative real-time PCR (RT-qPCR) analysis of PI3K gene expression in HepG2 cells treated with CSNPs-UPE, doxorubicin (DOX), and CSNPs-QPE. (a) CSNPs-UPE at 5 µg/mL induced a modest reduction of approximately 0.5-fold in PI3K expression. (b) DOX at 20 µg/mL resulted in nearly 80% inhibition of PI3K mRNA levels. (c) CSNPs-QPE at 5 and 20 µg/mL produced a pronounced downregulation, with up to 0.9-fold reduction in PI3K expression, demonstrating potent gene-silencing effects. Data are expressed as mean ± standard deviation (n = 3). Statistical significance: p < 0.05, p < 0.01, p < 0.001 versus control.

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Fig. 10.

(a-c) Half-maximal inhibitory concentration (IC₅₀) analysis of HepG2 cells treated with CSNPs-UPE, doxorubicin (DOX), and CSNPs-QPE. Dose–response curves show that CSNPs-QPE achieved a significantly lower IC₅₀ value (4.5 µg/mL) compared with the standard chemotherapeutic agent DOX (6.67 µg/mL) and the unloaded formulation CSNPs-UPE, indicating enhanced cytotoxic potency of the quercetin-loaded Pickering emulsion. Data represent mean ± standard deviation of three independent experiments.

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Fig. 11.

(a) Real-time PCR analysis revealed no significant fold change in AKT gene expression following treatment with CSNPs-UPE at a concentration of 20 µg/mL. (b) In contrast, doxorubicin (DOX) at a high concentration of 80 µg/mL produced a marked suppression of Akt expression, with >90% inhibition relative to the untreated control. (c) Notably, exposure of HepG2 cells to CSNPs-QPE at a low dose (5 µg/mL) resulted in >80% inhibition of AKT gene expression, demonstrating potent downregulation at sub-cytotoxic levels. Data are presented as mean ± standard deviation (SD) from three independent experiments. Statistical significance was determined relative to the control group (*p < 0.05, **p < 0.01, **p < 0.001).

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Fig. 12.

(a-c) Half-maximal inhibitory concentration (IC₅₀) analysis demonstrated that CSNPs-QPE exhibited a markedly lower IC₅₀ value (2.24 µg/mL) compared with the standard chemotherapeutic agent doxorubicin (3.43 µg/mL) and the unloaded formulation CSNPs-UPE. This finding indicates the superior cytotoxic efficacy of the quercetin-loaded Pickering emulsion relative to both the conventional drug and the unloaded control.

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Fig. 13.

Quantitative real-time PCR (RT-qPCR) analysis of mTOR gene expression in HepG2 cells following treatment with CSNPs-UPE, doxorubicin, and CSNPs-QPE. (a) CSNPs-UPE at 5 µg/mL induced only a 0.2-fold change in mTOR expression, indicating minimal inhibitory activity. (b) DOX at 80 µg/mL produced >70% suppression of mTOR mRNA levels. (c) CSNPs-QPE at 5 and 20 µg/mL achieved >90% inhibition of mTOR expression, demonstrating potent down-regulatory effects. Data are presented as mean ± standard deviation (n = 3). Statistical significance: p < 0.05, p < 0.01, p < 0.001 versus control.

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Fig. 14.

(a-c) Half-maximal inhibitory concentration (IC₅₀) analysis of HepG2 cells treated with CSNPs-UPE, doxorubicin, and CSNPs-QPE. Dose–response curves demonstrate that CSNPs-QPE achieved a significantly lower IC₅₀ value (2.24 µg/mL) compared with the standard chemotherapeutic agent DOX (10.01 µg/mL) and the unloaded formulation CSNPs-UPE, indicating superior cytotoxic potency of the quercetin-loaded Pickering emulsion. Data represent mean ± standard deviation of three independent experiments.

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4. Discussion

Nanodrug delivery systems facilitate the efficient transport of natural products as therapeutic agents. Such systems enhance drug release profiles in a sustained manner, reduce side effects, improve bioactivity, and enable targeted delivery (Islam et al., 2017). Quercetin is a potent bioactive compound present in various functional foods; however, its therapeutic use is limited due to poor water solubility, low gastrointestinal (GI) membrane permeability, and the need for high doses, which can lead to acute toxicity, kidney dysfunction, gastrointestinal disturbances, and allergic reactions (Andres et al., 2018). Nanoscale formulations of quercetin have been developed to address these limitations. In this study, quercetin-based Pickering emulsions were synthesized and evaluated for their anticancer activity against hepatic cancer cells. Pickering emulsions are particularly attractive as nanocarriers owing to their biodegradability, minimal toxicity, facile synthesis, chemical modifiability, and biocompatibility (Andres et al., 2018). Chitosan–tripolyphosphate (CS-TPP) nanoparticles were used as stabilizers to produce size-controlled Pickering emulsions with enhanced biological activity and improved stability.

Quercetin-loaded Pickering emulsions were prepared with minor modifications to previously reported protocols (Shah et al., 2016). The emulsions remained stable at room temperature for at least 6 weeks (±1.22), demonstrating greater stability as compared to several reported nanocarrier systems (ye et al., 2021). Nanoparticles possess unique biological utility due to their high surface charge, which promotes interactions with natural compounds. In this formulation, nanoparticles served as stabilizers at the oil-water interface of the Pickering emulsion. Medium-chain triglycerides (MCTs), specifically lauric acid, were used as the oil phase. Within the body, lauric acid is either incorporated into newly synthesized triglycerides that enter the lymphatic system or, more commonly, transported directly to the liver. The structural properties of coconut-derived triglycerides facilitate more efficient digestion compared to other long-chain triglycerides, such as palmitic acid. Consequently, lauric acid exhibits a high clearance rate with minimal side effects, making it an effective component for targeted drug delivery (Dayrit et al., 2015). The CSNP-stabilized Pickering system developed in this study incorporates two major advantages that have been repeatedly identified in the recent literature: (1) the intrinsic interfacial robustness of particle-stabilized emulsions, and (2) the functional benefits of chitosan nanoparticles as stabilizers. Recent reviews highlight that PEs reduce the need for synthetic surfactants, improve physical stability against coalescence, and can enhance the biological accessibility of hydrophobic actives in nutraceutical and pharmaceutical contexts (Wang et al., 2025). Recently, a study reported the synthesis of quercetin-loaded Pickering emulsions for tropical skin care applications in which chitosan nanoparticle was used as a solid stabilizer, which is in line with our study as well (Sainakham et al., 2025).

The nanoparticles exhibited a smaller size than the synthesized Pickering emulsions due to interactions with oil and the encapsulated drug. The size distribution results were consistent with the previously reported study (Shah et al., 2016). Controlled particle size is critical for efficient cellular uptake of CSNPs-QPE. UV–visible spectroscopy revealed absorption peaks at 378.8 nm and 341.9 nm for CSNPs-QPE and CSNPs-UPE, respectively. Quercetin alone displayed a characteristic peak at 375 nm, while nanoparticles exhibited a peak at 310 nm. The red shift from 375 to 378.8 nm indicates successful bonding between quercetin and the emulsion. FTIR analysis confirmed chemical interactions among emulsion components, with CSNPs-UPE showing characteristic absorption peaks at 2879, 2945, 916, 750, 1707, and 1286 cm⁻¹, whereas CSNPs-QPE exhibited a peak at 3304 cm⁻¹ corresponding to phenolic OH of quercetin and surface hydroxyls and N–H stretching from chitosan (Fig. 3b). The 1448 cm^-1 peak is attributed to aromatic C=C stretching / ring vibrations associated with phenolic groups of quercetin and its appearance in CSNPs-QPE suggesting the presence of quercetin at the interface. A peak at 1230 cm-1 is associated with C–O stretching / C–O–C vibrations (quercetin phenolic ether and lauric acid C–O) and phosphate-related vibrational modes from TPP–chitosan interactions (Fig. 3b). These data are consistent with previously reported findings (Kajbafvala et al., 2017), supporting the successful synthesis of the Pickering emulsions. A study conducted by Xu et al. also reported the broad absorption peak at 3400 cm-1 along with a peak at 1500 cm-1 depicting C–O stretching vibration (XU et al., 2019). FTIR results along with UV–visible spectra, suggesting multiple chemical modifications during emulsion formation.

Particle size distribution analysis revealed an average emulsion diameter of 238 nm with a surface charge of +59.5 mV. Nanoscale size (∼238 nm) allows efficient internalization by cancer cells through endocytosis. Such emulsions can penetrate biological barriers more effectively, such as tumor interstitial spaces, and can improve drug deposition at the target site. Further +59.5 mV zeta potential demonstrates strong electrostatic repulsion among droplets to avoid agglomeration during storage or biological circulation. Another benefit of positive surface charge involves the electrostatic attraction to negatively charged phospholipid membranes of cancer cells, promoting better cellular adhesion and uptake of the encapsulated quercetin. The polydispersity index (PDI) of 0.457 (<1) indicates a uniform size distribution. The high zeta potential of the quercetin-loaded emulsions suggests strong electrostatic interactions between the oil and aqueous phases (Shah et al., 2016). Fluorescence imaging confirmed quercetin encapsulation, as quercetin is naturally fluorescent (Yang et al., 2017). Transmitted light images revealed spherical Pickering emulsion particles with smooth boundaries (Figs. 5a and b), consistent with dynamic light scattering (zeta sizer) measurements. Scanning electron microscopy (SEM) further confirmed the formation of uniformly shaped, size-controlled Pickering emulsions (Fig. 5c), in agreement with the findings of Albalawi et al. (Albalawi et al., 2023).

The Pickering emulsions achieved a high quercetin encapsulation efficiency of 88.8% (±0.95), exceeding values typically observed in comparable nano formulations (Rodríguez‐Félix et al., 2019). High encapsulation efficiency enhances bioavailability and is often associated with smaller nanoparticle size (Rodríguez‐Félix et al., 2019). The therapeutic efficacy of nanodrug systems depends on controlled drug release following administration. Sustained release offers several advantages, including reduced dosing frequency, fewer adverse effects, and prolonged therapeutic action (Albalawi et al., 2023). In this study, quercetin release from the Pickering emulsions followed a diffusion-controlled pattern, with higher release observed at pH 4.0 compared to pH 7.4. This pH-dependent behavior suggests preferential drug release in the acidic microenvironment of cancer cells (Abdella et al., 2023).

Cytotoxicity of the synthesized nanoformulation was assessed in HepG2 liver cancer cells. At a concentration of 80 μg/mL, CSNPs-QPE induced >90% cell death (Fig. 7a). At lower concentrations (5 μg/mL), both CSNPs-QPE and doxorubicin achieved approximately 50% cell inhibition (Fig. 7a). The half-maximal inhibitory concentration (IC₅₀) for CSNPs-QPE was 20 μg/mL after 48 hours of treatment, reflecting the time required for sustained drug release from the nanoformulation (Fig. 7b). MTT assay results demonstrated a dose-dependent decrease in cell viability, with the highest viability observed at 2.5 μg/mL and progressively reduced survival at higher concentrations. These findings are consistent with previous reports demonstrating effective inhibition of tumor cell proliferation following the administration of betanin-encapsulated nanoparticles to breast cancer cells (Rehman et al., 2023).

A wound-healing (scratch) assay was performed to evaluate the impact of CSNPs-QPE and doxorubicin on 2D tumor cell migration, a critical step in metastasis involving cell movement through the extracellular matrix. The wound-healing assay revealed that CSNPs-QPE significantly suppressed the lateral migration of HepG2 cells in a two-dimensional environment, indicating strong inhibition of a key early step in tumor metastasis. Both tested concentrations (5 and 20 μg/mL) produced markedly greater inhibition of scratch closure compared with doxorubicin, underscoring the enhanced antimetastatic potential of the Pickering emulsion system. These results align with earlier reports that nanoparticle-based CuS@mSiO₂-PEG formulations impede cancer cell motility by disrupting cytoskeletal remodeling and adhesion signaling (Deng et al., 2018).

Recent research studies highlight the effective delivery of macromolecules by nanoparticles targeting multiple proteins involved in tumor progression (Dong et al., 2023; Das et al., 2024; Kapoor et al., 2025). Aberrant activation of the PI3K/Akt/mTOR signaling pathway is a key driver of hepatocellular carcinoma, promoting uncontrolled proliferation, angiogenesis, and metastasis while also contributing to chemoresistance (Sorice et al., 2014; Dong et al., 2021). The present study demonstrates that quercetin‐loaded chitosan Pickering emulsions (CSNPs-QPE) markedly downregulate the PI3K/Akt/mTOR signaling cascade in HepG2 cells, with more than 80% inhibition of PI3K and mTOR gene expression (Figs. 9 and 13) and significant suppression of Akt and mTOR even at low concentrations (5 μg/mL) (Figs. 11c and 13c) indicating potent antiproliferative effects. These results are consistent with earlier reports showing that quercetin can modulate PI3K/Akt/mTOR signaling to inhibit hepatocellular carcinoma (HCC) progression (Dong et al., 2021). Another study illustrated the antitumor activity of quercetin nanoparticles by inactivating the protein expression level of Akt and ERK1/2 signaling pathway in liver cancer cells (Zhang et al., 2025). Compared with these carriers, the CSNPs-QPE developed in this study achieved lower IC₅₀ values for PI3K, Akt, and mTOR inhibition than doxorubicin, indicating enhanced potency (Figs. 10, 12, and 14). In Fig. 10, lower IC₅₀ values of CSNPs-QPE (4.5 μg/mL) for inhibiting PI3K expression in comparison to doxorubicin (6.67 μg/mL) illustrate its greater potency. The same observations were recruited from analysis of IC₅₀ values of CSNPs-QPE for inhibiting Akt and mTOR expression pattern (Figs. 12 and 14). Particularly, IC₅₀ values of CSNPs-QPE (2.244 μg/mL) for inhibiting mTOR depicted more therapeutic potency in comparison to doxorubicin (10.01 μg/mL) (Fig. 14). This therapeutic effectivity may be attributed to the unique interfacial stabilization and high surface charge of Pickering emulsions, which can improve cellular uptake and sustained drug release relative to conventional liposomes or polymeric nanoparticles. Furthermore, the ability of CSNPs-QPE to significantly suppress mTOR expression at lower doses signifies their potential to overcome mTOR-driven chemoresistance, a major limitation of current HCC therapies. Quercetin primarily exerts its anticancer effects in HCC by downregulating P4HA2 and promoting Phosphatase and Tensin Homolog (PTEN) activation, thereby inhibiting the PI3K/Akt/mTOR signaling pathway. This mechanism positions quercetin as a promising therapeutic strategy against HCC (Zhang et al., 2025; Karabat et al., 2025). Based on these previous studies, we coupled the therapeutic efficacy of quercetin by incorporating it into Pickering emulsions to improve cellular uptake, increased intracellular retention and sustained release of drug. Taken together, these findings extend previous evidence on quercetin nanoformulations by demonstrating that chitosan-based Pickering emulsions offer an effective and biocompatible platform for targeted regulation of the PI3K/Akt/mTOR pathway.

5. Conclusions

Biodegradable quercetin-encapsulated chitosan Pickering emulsions exhibited significant in vitro cytotoxicity against human liver cancer cells, accompanied by inhibition of cell migration and downregulation of the PI3K/Akt/mTOR signaling pathway. The emulsions also demonstrated sustained quercetin release, indicating their potential as a controlled nano-drug delivery system. While these findings highlight CSNPs-QPE as a promising platform for quercetin delivery, the evidence is currently limited to in vitro experiments. Comprehensive preclinical studies and scale up evaluations are essential before any clinical translation.