Characterization and preliminary cytotoxic effects of pomegranate peel extract-loaded nanoparticles on HepG2 cells.

Introduction

Punica granatum (pomegranate), a member of the Punicaceae family, is a red and juicy fruit valued for its flavor and therapeutic properties1. Cultivated worldwide, it exhibits genetic and phytochemical diversity2. The fruit comprises the aril, the seed, and the peel. The peel, often-discarded byproduct, is rich in bioactive compounds, like polyphenols, flavonoids, dietary fibres, and organic acids, making it beneficial for medicinal and nutraceutical purposes3,4.
The extraction of these bioactive molecules from the peels has been optimised using solvents like methanol, ethanol, and water, which impacts both the yield and composition of the resulting pomegranate peel extract (PPE). PPE shows a range of biological activities, notably antioxidant, anti-inflammatory, antimicrobial5, antidiabetic6, and anticancer7 properties, besides positive effects on cardiovascular health8 and reproductive functions9 in animal models. However, the therapeutic potential of PPE is often hindered by low solubility, poor bioavailability, and instability. Nanoencapsulation technology offers a critical solution by improving the delivery and efficacy of plant-derived compounds4. This approach aligns with a highly sustainable trend in nanomedicine, where fruit peel extracts are used as a therapeutic payload and also as “green” agents for synthesizing novel nanomaterials with enhanced anticancer properties10, e.g., nanoparticles (NPs) derived from banana peels11 and orange peels12 have shown efficacy against liver and breast cancer cell lines, respectively. Chitosan (CS), a natural, biodegradable polymer, acts as an apt nanocarrier due to its mucoadhesive, protective, and antioxidant properties. Chitosan nanoparticles (CSNPs), often synthesized via the ionic gelation method, offer a high surface-area-to-volume ratio, enabling efficient loading and controlled release of encapsulated phytochemicals13. CSNPs successfully encapsulated various plant extracts, like green tea and cranberry, enhancing their therapeutic potential14.
The use of nanoencapsulated plant extracts for hepatocellular carcinoma (HCC) treatment remains a recent and underexplored area15. The human hepatocellular carcinoma cell line, HepG2, serves as a reliable in vitro model due to its physiological relevance, for evaluating the cytotoxicity of pharmacological agents16. And for preliminary high-throughput screening of anticancer compounds, the MTT assay is a sensitive, reproducible method, and a gold standard17.
Existing oncology research confirms the therapeutic potential of various PPEs and CS nano-carriers, individually or with other payloads18–21. The novelty of our work stems from their synergistic and unique combination of this specific CSPPE formulation against HCC. Therefore, this study aims to synthesize CSPPE NPs, investigate their new physicochemical characteristics, and quantify and compare the cytotoxic efficacy of aqueous PPE versus its CS-encapsulated nanoformulation (CSPPE) on HepG2 cells using the MTT assay. Our goal is to compare the efficacy of the native and nanoencapsulated PPE, providing a foundation for future HCC nanotherapy interventions.

Materials and methods

Preparation of pomegranate Peel extract (PPE)
Fresh pomegranate peels were collected from a reputable juice outlet in Sohag Governorate, Egypt, washed thoroughly, and stored at 4 °C until processing. Two kilograms of peels were immersed in 1 L of bi-distilled water and heated at 90–95 °C for 7 h. After cooling to room temperature, the mixture was filtered through a mesh sieve and Whatman No. 1 filter paper.
To determine the extract concentration, a 10 mL aliquot of the aqueous extract was freeze-dried to constant weight, yielding approximately 200 mg of dry extract per 10 mL, corresponding to a stock concentration of 20 mg/mL dry weight. Working solutions for nanoparticle synthesis and biological assays were prepared by diluting this stock with bi-distilled water to the required concentrations. Fresh PPE was used for all experiments, and the remaining stock was stored at 4 °C and used within 7 days to minimize degradation.

Synthesis of Chitosan nanoparticles loaded with PPE (CSPPE)
CSPPE nanoparticles were prepared using a modified ionic gelation method14 at the Nanotechnology Research and Synthesis Unit, AHRI, Assiut, Egypt.
Preparation of solutions:

PPE solution (20 mg/mL) was sonicated for 5 min and stirred at 3000 rpm at 25 °C until homogeneous. Tween-80 (150 µL, Polysorbate 80) was added dropwise to stabilize the mixture.

Chitosan solution was prepared by dissolving 0.2 g of low molecular weight chitosan (M.W. 1526.6; CAS No.: 9012-76-4; India) in 40 mL of 1% (v/v) acetic acid to obtain a 0.5% (w/v) solution. The solution was stirred overnight at 25 °C, then adjusted to pH 4.6 using 1 M NaOH, and filtered through a 0.45 μm syringe filter.

Nanoparticle formation:

PPE (5% w/w relative to chitosan mass, i.e., 10 mg PPE for 0.2 g CS) was added dropwise to the chitosan solution under stirring at 1500 rpm for 30 min.

TPP (0.2% w/v, CS: TPP mass ratio 4:1) was then added dropwise under continuous stirring at 1000 rpm for 20 min to induce ionic gelation.

The nanoparticle suspension was centrifuged at 15,000 rpm for 30 min, washed twice with deionized water, and resuspended in 10 mL of water.

DLS measurements and stability:

Hydrodynamic diameter, polydispersity index (PDI), and zeta potential were measured using a Malvern Zetasizer Nano ZS (UK) at 25 °C. The freshly prepared CSPPE NPs had an average size of 408.8 ± 98.8 nm, PDI of 0.125, and zeta potential of + 25 to + 35 mV.

For stability assessment, aliquots were stored at 4 °C and re-measured after 1, 3, and 7 days. No significant changes in size or zeta potential were observed, confirming that the nanoparticles remained stable for at least one week.

Characterization of CSPPE nanoparticles

Dynamic light scattering (DLS) and zeta potential
The hydrodynamic size, size distribution, and surface charge of the chitosan nanoparticles loaded with pomegranate peel extract (CSPPE) were evaluated using a Zeta-sizer (Model 3000 HS, Malvern Instruments, UK) at the Nanotechnology Research Unit, Faculty of Pharmacy, Al-Azhar University, Assiut.
For DLS measurements, an aliquot of freshly prepared CSPPE suspension was diluted 1:10 with deionized water to avoid multiple scattering effects and transferred to a disposable cuvette. Measurements were performed at 25 °C with a fixed scattering angle of 90° and a refractive index of 1.33 for water. The Z-average hydrodynamic diameter and polydispersity index (PDI) were calculated from intensity-weighted size distributions, providing insights into particle uniformity and aggregation tendency. Each measurement was repeated in triplicate to ensure reproducibility. Zeta potential measurements were conducted on the same suspension to assess surface charge and predict colloidal stability. Samples were placed in a folded capillary cell, and electrophoretic mobility was measured using the Smoluchowski model. Positive zeta potential values were expected due to the protonated amino groups of chitosan, and any changes after PPE loading were interpreted as partial surface charge neutralization by the encapsulated polyphenols.

Transmission electron microscopy (TEM)
The morphology, size, and structural characteristics of CSPPE nanoparticles were examined using a transmission electron microscope (JEOL-100CX II, Japan) at the Electron Microscopy Unit, Assiut University, Assiut, Egypt.
For sample preparation, a drop of the CSPPE suspension was placed onto a carbon-coated copper grid and allowed to adsorb for 2–3 min. Excess liquid was gently removed using filter paper, and the grid was negatively stained with 2% (w/v) phosphotungstic acid for 30 s to enhance contrast. After air-drying at room temperature, the grids were observed under TEM at an accelerating voltage of 80–100 kV. Images were captured at multiple magnifications to visualize particle shape, size, and dispersion. The average particle diameter was determined by measuring at least 50 nanoparticles using ImageJ software. Morphological features such as sphericity, aggregation, and surface smoothness were qualitatively assessed.

Fourier-transform infrared spectroscopy (FTIR)
FTIR spectroscopy was performed using a NICOLET iS10 spectrometer (Thermo Fisher Scientific, USA) at the Chemistry Department, Faculty of Science, Assiut University, to investigate functional groups and potential molecular interactions in PPE and CSPPE nanoparticles.
Samples were prepared by mixing the dried powders of PPE and CSPPE with potassium bromide (KBr) at a 1:100 ratio, followed by thorough grinding to obtain a homogeneous mixture. The mixture was then compressed into transparent pellets under high pressure for spectral measurement. Spectra were recorded in the range of 4000–400 cm⁻¹ at a resolution of 4 cm⁻¹, with 32 scans per sample to enhance signal-to-noise ratio. The characteristic absorption bands were analyzed to identify hydroxyl (-OH), carbonyl (C = O), aliphatic C-H, aromatic C = C, and C-O functional groups. Comparisons between PPE and CSPPE spectra were conducted to detect any shifts, disappearance, or intensity changes of peaks, which could indicate successful encapsulation, hydrogen bonding, or other molecular interactions between the extract and the chitosan nanoparticle matrix.

Gas chromatography–mass spectrometry (GC–MS)
The volatile and semi-volatile chemical constituents of PPE and CSPPE were analyzed at the Laboratory of Functional Foods, Department of Dairy Sciences and Technology, Faculty of Agriculture, Alexandria University.
Sample preparation:

PPE: The aqueous extract was diluted 1:1 (v/v) with HPLC-grade methanol and filtered through a 0.45 μm PTFE syringe filter before GC–MS analysis. No derivatization steps (e.g., silylation or methylation) were applied, so the detected compounds reflect the native volatile/semi-volatile profile of the extract.

CSPPE: Lyophilized nanoparticles were dissolved in methanol and sonicated for 10 min to release encapsulated compounds. The suspension was centrifuged at 12,000 rpm for 10 min to remove insoluble chitosan residues. The supernatant was filtered through a 0.22 μm PTFE syringe filter prior to GC–MS injection. Again, no derivatization was applied.

Analyses were performed using a GC-TSQ mass spectrometer (Thermo Scientific, USA) equipped with a TG–5MS capillary column (30 m × 0.25 mm × 0.25 μm), following parameters described by El Bohi et al.22. Component identification was carried out by comparing mass spectra with WILEY 09 and NIST14 libraries.
GC–MS primarily detects volatile and semi-volatile compounds; therefore, non-volatile bioactives such as polyphenols, flavonoids, dietary fibers, and organic acids, abundant in pomegranate peel, are not fully captured by this method. The detected methyl esters (e.g., methyl oleate, methyl-CLA) likely reflect either naturally occurring fatty acid esters in the peel or partial esterification during sample handling, but no chemical derivatization was intentionally performed. Consequently, these methyl esters should be interpreted cautiously in mechanistic discussions, as fatty acids and phenolic compounds exhibit different anticancer properties. For comprehensive profiling of polyphenolic and other non-volatile compounds, complementary techniques such as HPLC or LC–MS are recommended23,24.

Cell cytotoxicity assay

Cell culture
The human hepatocellular carcinoma cell line HepG2 was obtained from Vacsera, Cairo, Egypt. Cells were maintained in RPMI 1640 medium (Gibco™, NY, USA; Cat. No. 11875093) supplemented with: 11.111 mM glucose, 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 0.025% Trypsin-EDTA. Cells were cultured in a humidified incubator at 37 °C with 5% CO₂ and passaged every 2–3 days to maintain logarithmic growth. Cell density was monitored using a hemocytometer and only cultures with > 95% viability (trypan blue exclusion) were used for experiments.

MTT cytotoxicity assay
The cytotoxicity of PPE, CSPPE, and blank chitosan nanoparticles (CSNPs, prepared without PPE) was evaluated using the MTT assay25–27 at Science Way Laboratory, Cairo, Egypt. All MTT assays were performed in three independent biological experiments, each with triplicate technical replicates.
Preparation of Samples and Controls:

PPE stock: The aqueous extract of pomegranate peel was freeze-dried to determine dry weight. The resulting concentration of the stock solution was ~ 20 mg/mL dry extract. Working solutions were freshly prepared by diluting stock in RPMI 1640 medium with 2% FBS to the desired concentrations.

CSPPE nanoparticles: The PPE content in the nanoparticles was quantified via UV–Vis spectroscopy (λ_max 280 nm). Encapsulation efficiency (EE%) and loading capacity (LC%) were determined: EE% = 85.3 ± 2.1%, LC% = 4.2 ± 0.3%. Reported CSPPE concentrations correspond to PPE equivalents, not total NP mass.

Blank CSNPs: Chitosan nanoparticles prepared under identical conditions without PPE were used as a control to distinguish effects of the nanoparticle matrix or residual reagents from PPE cytotoxicity.

Vehicle control: RPMI 1640 with 2% FBS, no nanoparticles or extract.

Assay Procedure:

HepG2 cells were seeded into 96-well plates at a density of 1 × 10⁴ cells/well and allowed to attach for 24 h.

Two-fold serial dilutions of PPE and CSPPE were prepared, starting at 1000 µg/mL PPE equivalents down to 1.95 µg/mL across 10 concentrations. Blank CSNPs were tested at equivalent NP concentrations.

Each treatment and control was applied in triplicate wells, and each experiment was repeated three times.

Plates were incubated for 24 h at 37 °C, 5% CO₂. Cell morphology was observed under a phase-contrast microscope (Zeiss, Germany) and compared to untreated and blank CSNP controls.

MTT reagent (20 µL, 5 mg/mL) was added to each well, plates were shaken at 150 rpm for 5 min and incubated for 4 h at 37 °C. Formazan crystals were solubilized with 200 µL DMSO and shaken for 10 min to ensure complete dissolution.

Absorbance was measured at 560 nm with background correction at 620 nm using a Biotek ELx800 microplate reader.

Cell viability (%) was calculated relative to vehicle control:

Cytotoxicity classification followed Mosmann28: 90%: non-cytotoxic, 90–60%: Slightly cytotoxic, 59–30%: Moderately cytotoxic, and < 30%: Strongly cytotoxic.

Rationale for Controls:

Blank CSNPs confirmed that the observed cytotoxicity is attributable to PPE payload and not the chitosan matrix or residual reagents.

Vehicle control allowed normalization of cell viability.

Data analysis
NP size distribution from TEM images was quantified using ImageJ software in conjunction with Origin (Origin Lab Corporation, Northampton, MA, USA), according to Zhang and Wang29. MTT assay experiments were conducted in triplicate (n = 3) for optical density (O.D) measurements, as confirmed in the raw data presented in Table 1. The relationship between concentration and cell viability was plotted, and IC₅₀ values were determined using Origin software. This determination involved fitting the concentration-response data to a non-linear regression curve (sigmoidal dose–response, variable slope) to accurately derive the IC₅₀ value and its associated SD, as shown in Table 1. The 95% confidence intervals were also derived for each IC₅₀ value. To compare IC₅₀ values, an independent two-sample t-test was performed using GraphPad Prism 7. A p-value ≤ 0.05 was considered statistically significant. All reported data are expressed as mean ± SD.

Results

Characterization of synthesized CSPPE

Dynamic light scattering (DLS) and zeta potential measurements
The hydrodynamic properties and colloidal stability of CSPPE nanoparticles were characterized using Dynamic Light Scattering (DLS) and zeta potential analysis. DLS measurements revealed a Z-average diameter of 408.76 ± 98.75 nm with a low polydispersity index (PDI) of 0.125, indicating a relatively uniform nanoparticle population. The intensity-based size distribution curve (Fig. 1) displayed a bell-shaped profile, with the majority of nanoparticles ranging between 100 and 500 nm. The sharp peak reflects the dominant particle size contributing to the scattered light intensity, while the narrow distribution and rapid decline on either side suggest minimal presence of significantly smaller or larger particles, confirming sample homogeneity.

The relatively large hydrodynamic size compared to the core size observed by TEM (51.5 ± 12.6 nm, n = 50) is expected, as DLS measures nanoparticles in their hydrated state, including solvation layers and possible polymer swelling, whereas TEM visualizes dehydrated cores. Number- and volume-based DLS distributions were analyzed, showing that the majority of nanoparticles cluster around smaller sizes than the intensity-weighted Z-average, partially explaining the discrepancy. Zeta potential measurements were conducted to evaluate surface charge and predict long-term stability. Unloaded CSNPs exhibited a positive surface charge of approximately + 25–45 mV, consistent with the presence of protonated amino groups in chitosan. Loading with PPE led to a moderate decrease in zeta potential to approximately + 15–35 mV, likely due to partial neutralization of surface charges by phenolic constituents. Despite this reduction, the nanoparticles retained sufficient positive charge to ensure electrostatic stabilization, indicating good colloidal stability and low tendency for aggregation over time.

Transmission electron microscopy (TEM) analysis
TEM images (Fig. 2) visually confirm that the CSPPE NPs are successfully synthesised within the nanometer scale, as denoted by the scale bars. The NPs exhibit a generally uniform size distribution, reflecting precise control during synthesis. Morphologically, the NPs are primarily spherical, with minor deviations such as asymmetry or faceting in some instances. The NPs appear well dispersed across the field of view with only minimal aggregation, and most exist as isolated entities rather than clusters, suggesting that attractive interparticle interactions were effectively minimized. The absence of extensive agglomeration further supports the effective dispersion and stability of the formulation. For quantitative analysis of NPs size distribution (Fig. 3), the methodology of Zhang and Wang29 was applied.

Fourier-Transform infrared (FTIR) spectroscopy analysis
FTIR spectroscopy showed the chemical composition of raw PPE and CSPPE NPs. The spectrum (Fig. 4) was analyzed across four main regions related to distinct functional group vibrations:

Region 1 (4000–2500 cm-1): A broad peak at ~ 3490 cm⁻¹ in both PPE and CSPPE spectra correlates to hydroxyl (–OH) groups, likely from alcohols, phenols, or carboxylic acids. It is more intense in CSPPE, suggesting a higher –OH content. Besides, a peak at ~ 2950 cm⁻¹, indicative of aliphatic C–H stretching in both samples, with greater intensity in CSPPE.

Region 2 (2500–2000
cm-1): It showed no significant peaks, indicating the lack of triple bonds.

Region 3 (2000–1500
cm-1): A strong band at ~ 1740 cm⁻¹, owing to carbonyl (C = O) stretching (carboxylic acids, esters, or amides), is more prominent in PPE, suggesting higher carbonyl content. A peak near 1610 cm⁻¹ corresponds to aromatic C = C bonds, with slightly higher intensity in PPE.

Region 4 (1500–500
cm-1): A strong peak at ~ 1120 cm⁻¹, indicating C–O bond stretching, is slightly more intense in PPE, reflecting a higher concentration of these functional groups.

GC–MS analysis

GC–MS of PPE
The chromatogram of PPE (Fig. 5) identified 22 compounds, accounting for 100% of the extract (Table 2). The major constituents that exist at the highest proportions were methyl oleate (40.94%), methyl 9-cis,11-trans-octadecadienoate (20.7%), and oleic acid (11.19%), with retention times (RTs) ranging from 7.65 to 29.57 min, and are likely the major bioactive components (Fig. 6). Minor constituents, including palmitic acid, 4 H-pyran-4-one derivatives, dodecanoic acid esters, and octinoxate, among others, each present at < 5%. However, they may contribute synergistically to the extract’s biological activity.

The chromatogram of CSPPE (Fig. 7) defined 10 compounds comprising 99.99% of the extract (Table 3). The major compounds were methyl 11-octadecenoate (38.96%), oleic acid (20.70%), and methyl 9-cis,11-trans-octadecadienoate (19.88%) (Fig. 8). Other constituents included hexadecanoic acid, methyl esters, and arabinitol pentaacetate. The least abundant was 11-octadecenal (0.79%).

The GC–MS chromatogram of PPE displayed dual peaks corresponding to oleic acid, which likely represent cis- and trans-isomers or positional isomers. Such isomeric diversity is commonly observed in plant-derived extracts and can arise either naturally or during derivatization steps. These isomers may differ in their biological activities, including cytotoxic effects, and therefore contribute collectively to the observed bioactivity of the extract. Recognizing the presence of these isomers provides a more accurate interpretation of the chemical profile and helps in understanding the potential mechanisms underlying the enhanced cytotoxicity of CSPPE.

Entrapment and loading efficiency
The encapsulation efficiency (EE%) and loading capacity (LC%) of PPE within the CSPPE nanoparticles were determined using UV–Vis spectroscopy. The results showed an EE% of 85.3 ± 2.1% and an LC% of 4.2 ± 0.3%, confirming effective incorporation of PPE. These data indicate that the nanoparticles deliver a precise amount of extract per unit mass, ensuring that biological assays reflect the activity of PPE rather than total nanoparticle mass.

Cell cytotoxicity assay

Quantitative assessment
The MTT assay results on HepG2 cells are summarized in Table 4; Figs. 9 and 10, presented as cell viability (%).

Raw PPE: Slight effects on cell viability were observed at 1000 and 500 µg/mL, with viability of 43.92 ± 0.82% and 81.95 ± 1.15%, respectively. At lower concentrations (250–31.25 µg/mL), viability remained above 90%, indicating minimal cytotoxicity. The IC₅₀ of raw PPE was 921.8 ± 18.6 µg/mL.

CSPPE (PPE-equivalent concentration): CSPPE significantly reduced cell viability at all tested concentrations, with values ranging from 0.02% to 99.80%, demonstrating markedly stronger inhibitory effects compared to raw PPE. The IC₅₀ was 12.38 ± 0.28 µg/mL PPE equivalent, confirming ~ 75-fold higher potency.

Blank CSNPs: Cells treated with chitosan nanoparticles without PPE showed > 90% viability across all tested concentrations.

Qualitative evaluation via light microscopy
Microscopic observations (Figs. 11 and 12) confirmed the MTT assay findings: (a) Control cells and blank CSNPs: High confluence and normal morphology, (b) PPE-treated cells: Concentration-dependent changes, including reduced cell density, loss of adhesion, and rounding/shrinkage at higher concentrations. Lower doses produced minimal morphological changes, (c) CSPPE-treated cells: More pronounced cytotoxic features even at moderate doses. High-dose exposure caused significant shrinkage, rounding, and detachment. At 125 µg/mL (PPE-equivalent), cytotoxicity was still apparent but reduced, while at 31.25 µg/mL, morphology resembled control.

Discussion
The study used the ionic gelation method for CSPPE NPs synthesis, an affordable and eco-friendly technique based on electrostatic interactions between cationic polymer CS and anionic crosslinker TPP. This method enhances stability and reduces chemical toxicity by avoiding harsh conditions and organic solvents30. CS and TPP are safe and biocompatible, making them ideal for drug delivery systems31. CS degradation products are generally accepted as non-toxic, supporting CS use in formulating CSPPE NPs. CS NPs are considered low in toxicity in acute studies, but factors like concentration, size, and exposure duration can affect on cell viability and cause organ damage in certain contexts32. After synthesis, characterising the size and shape of NPs is crucial as they directly impact nanocarrier functionality. The most prevalent methods for NPs analysis are DLS and TEM33.
The Z-average diameter by DLS was 408.76 ± 98.75 nm, with a low PDI of 0.125. Although synthesized via ionic gelation (which often yields particles over 100 nm), the PDI value ≤ 0.5 confirms the CSPPE suspension has good colloidal stability and high monodispersity30,34. This homogeneity is also supported by the narrow width and bell-shaped intensity curve (Fig. 1), showing a uniform NPs population that did not tend to aggregate or clump together over a short period. Whereas high-resolution TEM images (Fig. 2) confirmed that CSPPE NPs were synthesized within the nanometre scale range. The majority of NPs were spherical and appeared with no marked clusters or extended agglomerates across the field of view, suggesting effective dispersion and stability. Further, analysis of 50 individual NPs from TEM images using ImageJ software revealed an average size of 51.54 ± 12.58 nm. These findings together indicate the successful synthesis of uniform, stable, and well-dispersed CSPPE NPs. This size discrepancy, where DLS reports significantly larger sizes than electron microscopy (408.76 ± 98.75 vs. 51.54 ± 12.58 nm), is consistent across various studies on different NPs. Soltanzadeh et al.14 found that SEM images of unloaded CSNPs and PPE-loaded CSNPs showed a spherical morphology and homogeneous size distributions, with mean sizes of 90.6 ± 21.5 and 127.3 ± 38.7 nm, calculated by ImageJ software based on statistical analysis of 100 and 46 NPs, respectively. This suggests that PPE incorporation into CSNPs increases the mean particle diameter. Likewise, the hydrodynamic sizes by DLS of chemically and green-synthesised titanium dioxide NPs were 319 and 224 nm, respectively, while the TEM image revealed sizes of nearly 5 and 27 nm, respectively35.
This size discrepancy arises from the principles of the two techniques: (1) DLS measures the particles in a hydrated suspension, yielding the “hydrodynamic size”, which includes the NPs’ core plus the surrounding layer of adsorbed solvent molecules or stabilizers, resulting in a significantly larger diameter36. Conversely, TEM measures the particle in a dry state, which often leads to its shrinkage and a smaller measured size34. (2) DLS computes the Z-average using “intensity distribution”, which overrepresents any aggregates or larger particles in a sample - even at low concentrations- due to their stronger light-scattering properties, skewing the average size toward larger values, obscuring smaller, more numerous particles, so the peak of the size distribution curve in DLS can be misleading. Hydrodynamic radii can be much larger -sometimes up to tenfold- than the actual sizes measured by TEM33.
The NPs’ size is vital for their pharmacokinetics, influencing their cellular uptake, biodistribution, biological activity and systemic toxicity, hence their therapeutic action37. Hydrodynamic size (measured by DLS) is more biologically relevant in a fluid medium. Optimal sizes (15–100 nm) ensure longer circulation and better access to targeted tissue, whereas larger ones are quickly cleared by phagocytic cells and build up in the liver and spleen. Very small NPs (< 10–15 nm) are eliminated by the kidneys38. Usually, smaller NPs have higher cellular uptake and cytotoxicity, yet this varies with the cell type and its common uptake pathway39. This size-dependent behavior also governs NPs’ penetration in tumor spheroid models40. This study validates successful uptake with a 75-fold lower IC50 compared to the raw extract, confirming favourable size characteristics for HepG2 internalisation. In biological environments like blood plasma, NPs adsorb proteins and other biomolecules, forming a dynamic “biomolecular corona”. Its formation is affected by the NPs’ physicochemical properties, including size, and alters their biological identity, serving as a “camouflage”. While NPs allow biological entities, enhancing their biocompatibility and prolonging their circulation time, it also modifies their interactions with cells and the immune system, influencing the therapeutic effectiveness41.
The FTIR spectra of unloaded CSNPs and PPE-loaded CSNPs (CSPPE) were compared to evaluate possible chemical interactions upon loading. Both spectra displayed the characteristic functional groups of chitosan and PPE, including hydroxyl (-OH), aliphatic C–H, carbonyl (C = O), carbon–carbon double bond (C = C), and C–O stretches. No significant spectral shifts were observed between unloaded CSNPs and CSPPE, indicating that the primary chemical structure of PPE and chitosan was preserved during encapsulation. However, differences in peak intensity were evident: (a) CSPPE showed increased intensity of the -OH, aliphatic C–H, and C = O bands, suggesting successful incorporation and physical entrapment of PPE within the CSNP matrix, (b) Conversely, raw PPE displayed slightly more prominent C–O and C = C stretches. These intensity changes, rather than the appearance of new peaks or disappearance of existing ones, indicate that PPE is physically encapsulated without covalent modification of the polymer or the extract. The observations align with previously reported FTIR profiles of PPE, confirming the presence of N–H and O–H bands, C–H, C = O, aromatic ring vibrations, and C–O stretching of carboxylic acids14.
The GC-MS analysis of PPE (Table 2) and CSPPE (Table 3) revealed a significant decrease in the number of chemical constituents by nearly half, from 22 compounds in PPE to only 10 in CSPPE, although seven compounds remained common to both. The predominant compounds were identical in both PPE and CSPPE, based on their peak area (%), detected at almost the same RTs, but their concentrations (area%) varied slightly. This reduction aligns with observations in other nano-emulsion studies42,43, which also noted a decrease in the volatile compounds using GC-MS analysis. The absence of some compounds after nanoencapsulation is mainly due to: (1) nanoencapsulation protects minor ingredients from rapid volatilization or degradation, caused by environmental factors like light, oxygen, moisture, and pH changes, thus enhancing stability and shelf life. The CS matrix serves as a physical barrier, effectively achieving encapsulation goals44. Notably, the main active compounds in both PPE and CSPPE remained unchanged, suggesting the successful retention. (2) Analytical limitations of GC-MS, which ideally separates volatile and semi-volatile organic compounds without decomposition, but the harsh conditions of the GC-MS, like high temperatures and interactions with the stationary phase, can lead to degradation or chemical reactions of non-volatile and thermally unstable substances45. Also, strong interactions between the compounds and the encapsulation matrix can hinder their release, making them less detectable46. Thereby, this reduction is interpreted as a sign of effective physical entrapment rather than chemical loss, supported by the MTT assay results, which demonstrate a 75-fold lower IC50 against HepG2 cells compared to the raw extract, indicating better stability and therapeutic action of the NPs.
The chromatograms indicate that PPE and CSPPE are rich in beneficial fatty acids (FAs) and derivatives, contributing to their noted cytotoxicity in the MTT assay. The five most abundant components, shown in (Table 4), included: (1) [9-Octadecenoic acid (Z)-, methyl ester] is a monounsaturated omega-9 FA with anti-inflammatory and anti-androgenic activities, likely helpful for managing benign prostatic hyperplasia or androgen-dependent cancers47. (2) [methyl 9-cis, 11-trans-octadecadienoate] is an FDA-approved polyunsaturated FA for its anti-cancer, anti-inflammatory, and immune-boosting properties48. (3) Oleic acid impacts cell membrane fluidity, gene expression, regulates antioxidant enzyme synthesis and activity, with anti-inflammatory effects49. Notably, oleic acid showed dual peaks in GC-MS analysis at two different RTs, exactly, 29.57 and 30.09 s for PPE, and 29.58 and 30.09 s for CSPPE. The RT is a feature of each compound under certain analytical conditions; it is not unique to a single substance50. Also, the dual peaks are likely due to multiple isomers of modified FAs that can yield similar RTs in analysis51. (4) Octadecanoic acid, methyl ester, is found in vegetable and animal fats52 and is associated with reduced levels of inflammatory mediators and rheumatoid factor when delivered in quercetin-loaded nanoparticles53. (5) Hexadecanoic acid is a promising anti-tumour agent54, while its methyl ester derivative has anti-inflammatory and antifibrotic effects55.
Besides, the PPE chromatogram (Table 2) contained other valuable compounds, such as 3% [2,3-Dihydro-3,5-dihydroxy-6-methyl-4 h-pyran-4-one (DDMP)], a potent antioxidant56,57, 2.23% [Dodecanoic acid, 2,3-bis (acetyloxy) propyl ester], is a healthy medium-chain SFA with anticancer, antioxidant, and immune boosting properties58, and 1.11% [D-fructose derivatives] shows antitumor activity59.
The MTT assay evaluated the anti-proliferative effects of PPE and CSPPE on the HepG2 cell line in vitro. Quantitatively, both extracts showed concentration-dependent cytotoxicity, with declining cell viability as concentration increased. The IC50 for the raw PPE was 921.81 ± 18.61 µg/ml, while CSPPE had a significantly lower IC50 of 12.38 ± 0.28 µg/ml after 24 h (Table 1), indicating the nano-encapsulation of PPE significantly (p-value ≤ 0.0001) augmented its anticancer efficacy about 75-fold. Recent studies on nanoparticle-mediated delivery in hepatocellular carcinoma models60,61 have demonstrated that nano-encapsulation can enhance cytotoxicity and improve bioavailability of natural extracts. Compared to these reports, our CSPPE system shows superior loading efficiency and a ~ 75-fold reduction in IC₅₀, highlighting its potential as an effective HCC-targeted delivery platform. Also, the observed ~ 75-fold reduction in IC₅₀ of CSPPE compared to raw PPE demonstrates a pronounced enhancement of cytotoxicity against HepG2 cells. While such a large potency gain may appear extraordinary, it is consistent with previous reports on nanoparticle-mediated delivery of bioactive compounds, where improved cellular uptake, protection from degradation, and sustained release amplify biological effects37,39. To ensure that this effect is attributable to the encapsulated PPE rather than the delivery system itself, appropriate controls were incorporated: blank chitosan/TPP nanoparticles (without PPE) were tested and showed minimal cytotoxicity, confirming that the polymer matrix does not significantly affect cell viability. Similarly, the low concentration of Tween-80 used during preparation was verified to be non-toxic, and no interference of NPs with the MTT assay was observed, as confirmed by consistent absorbance readings in vehicle-only controls. Taken together, these results indicate that the observed cytotoxicity enhancement is indeed due to the encapsulated PPE and reflects the favorable physicochemical properties of the CSPPE nanoparticles, including size, surface charge, and efficient payload delivery.
Our findings support the anti-cancer potential of Punica granatum extracts against HepG2 and other human cancer cell lines. Ethanolic extracts from pomegranate peel and seed achieved IC50 values of 7.8 µg/ml versus 1.95 µg/ml, respectively, with the latter being significantly more toxic62. Also, different PPE concentrations (1000, 500, 250, 125, 62.5, and 31.25 µg/ml), prepared using pomegranate powder extracted from the peels and sterile saline, showed a dose-dependent inhibition of HepG2 proliferation at 24 h by the MTT assay, with an IC50 of almost 83.9 µg/ml, enhanced sensitivity to gamma irradiation63. As well, PPE-ZN NPs achieved an IC50 of 266.68 ± 23.11 µg/ml, compared to 657.47 ± 13.84 µg/ml for free PPE on HepG2 cells, indicating about 2.5-fold reduction, and a promising green strategy against liver cancer19. Conversely, various ethanolic PPE concentrations (37.5, 75, 150, and 300 µg/ml) showed no significant cytotoxicity on HepG2 cells after 24 h; however, 300 µg/ml decreased cell viability to 89.46% after 48 h64.
The alcoholic and aqueous PPE extracts affected differently on the proliferation of three human cancer cell lines, namely, prostate adenocarcinoma (PC3), ovarian cancer (SKOV-3), and colorectal carcinoma (HCT 116), as well as normal human fibroblasts (MRC-5). The methanol extract significantly hindered PC3 and SKOV-3 proliferation, followed by the aqueous extract at room temperature. The antiproliferative action on PC3 and HCT116 was concentration-dependent, with PC3 being notably sensitive, while SKOV-3 maintained growth similar to control cells. Importantly, normal fibroblasts remained largely unaffected by all the PPEs, as indicated by non-toxic IC50 values3. Notably, alcoholic PPE typically reported lower IC50 values than our aqueous PPE (921.81 ± 18.61 µg/ml), showing a stronger cytotoxic effect, as higher IC50 values reflect reduced toxicity65. Variations in the IC50 values of a drug on a specific cell line across studies indicate differing potencies due to several factors and can be classified as (i) biological factors (inherent variations to the tested cell lines), (ii) non-biological factors like the types and sources of culture reagents, and (iii) human factors arising from differences in experimental techniques and protocols66. This enhancement aligns with studies showing that nano-encapsulated formulations of natural compounds like boswellic acids, curcumin, and naringenin have significantly lower IC50 values against HepG2 cells after 24 and 48 h, compared to their free forms67.
The enhanced toxicity can be due to the synergistic effects of various factors, including the richness of PPE with bioactive ingredients with antioxidants, anti-inflammatory, and anti-cancer activities. In addition, nano-encapsulation delivery system improves the solubility and bioavailability of compounds, facilitating their passage through biological barriers, reducing degradation, extending circulation time and improving cellular uptake and targeted delivery, thereby boosting therapeutic effects68,69. Further, the CS matrix protects active phytochemicals from rapid degradation, acts as an antioxidant, and inhibits ROS generation. When combined with drugs, CS modifies the pharmacokinetics, boosts biodegradability and biocompatibility, and reduces side effects and toxicity70.

Limitations
This study has several notable limitations that should be considered when interpreting the results. First, an in-vitro release study of pomegranate peel extract (PPE) from the nanoparticles (CSPPE) was not conducted due to current experimental and analytical constraints. Without release kinetics data, it is difficult to fully understand the rate and extent of PPE liberation from the nanoparticles, which may directly influence the observed cytotoxicity. Future work will include detailed release profiling under physiological conditions to better correlate nanoparticle behavior with biological activity. Second, while the combination of the MTT assay and light microscopy provided preliminary evidence for the markedly enhanced cytotoxicity of CSPPE compared with raw PPE, mechanistic studies to differentiate apoptosis from necrosis were not performed. Understanding the precise mode of cell death is critical for evaluating therapeutic potential and safety. Subsequent studies will include a comprehensive panel of assays, including Annexin V/PI flow cytometry for apoptosis and necrosis discrimination, caspase-3 activation as an apoptotic marker, reactive oxygen species (ROS) generation analysis, mitochondrial membrane potential assessments, and TUNEL staining. These in vitro assays will be complemented by in vivo evaluations to assess systemic toxicity and therapeutic efficacy in relevant animal models. Finally, although CSPPE demonstrated a ~ 75-fold lower IC₅₀ against HepG2 cells compared with raw PPE, its selectivity toward non-cancerous hepatocytes has not yet been evaluated. Determining IC₅₀ values in normal hepatocytes, such as THLE-2 cells or primary hepatocytes, and calculating the selectivity index (SI) will be crucial to confirm the therapeutic window and safety profile of CSPPE. Addressing these limitations in future studies will provide a more comprehensive understanding of CSPPE’s bioactivity, mechanisms of action, and translational potential as an anticancer agent.

Conclusion
This study successfully synthesized chitosan/sodium tripolyphosphate nanoparticles encapsulating pomegranate peel extract (CSPPE NPs) using an eco-friendly ionic gelation method. Characterization by DLS and TEM confirmed that the nanoparticles possessed desirable physicochemical properties, including nanoscale dimensions, uniform size distribution (PDI < 0.5), and predominantly spherical morphology. While some discrepancies were observed between DLS and TEM measurements, a common occurrence due to differences in measurement principles, both techniques supported the formation of well-dispersed and stable nanoparticles. FTIR analyses verified the physical encapsulation of PPE within the CSNP matrix without altering its chemical structure. GC–MS revealed a partial reduction in detectable bioactive compounds post-encapsulation; however, the major constituents largely retained their relative concentrations, highlighting the effectiveness of CSNPs as carriers for bioactive delivery.
The novelty of this work lies in the pronounced enhancement of biological activity following nanoencapsulation. CSPPE NPs exhibited approximately a 75-fold lower IC₅₀ against HepG2 cells compared to raw PPE, indicating that the formulation not only preserves bioactive compounds but also enhances cellular uptake. This suggests that therapeutic effects may be achievable at lower doses in vivo, positioning CSPPE NPs as a promising anti-proliferative system with improved therapeutic index and potentially reduced systemic toxicity. Despite these encouraging in vitro results, this study is limited by its reliance on the MTT assay and a single cancer cell line (HepG2). Further investigations are warranted to elucidate the mechanisms of action, evaluate selective toxicity across normal and cancerous cells, and conduct in vivo studies to confirm safety, pharmacokinetics, and therapeutic efficacy.