Structural characterization and bioaccessibility of nanoliposomal lutein from pumpkin extract

Abstract

Pumpkin processing by-products represent a sustainable source of lutein; however, the poor stability and low gastrointestinal bioaccessibility of free lutein limit its practical application in functional foods. Previous studies have largely focused on either extraction optimization or encapsulation separately, lacking an integrated approach that combines green extraction with nanoencapsulation to address both recovery and delivery challenges. The objective of this study was to recover a lutein-rich carotenoid extract from pumpkin peel using supercritical CO₂ and subsequently encapsulate it into food-grade nanoliposomes (NLPs) at varying lutein/lecithin ratios (0.5%, 0.75%, 1.0%, and 1.5% w/w). The physicochemical properties, structural characteristics, antioxidant activity, in vitro release, and gastrointestinal bioaccessibility of the resulting formulations were systematically evaluated. Lutein-loaded NLPs exhibited mean particle sizes ranging from 109 to 272 nm with negative zeta potentials (− 22.1 to − 29.1 mV), indicating good colloidal stability. Encapsulation efficiency increased with lutein/lecithin ratio, reaching a maximum of 86.3% at 1.0% loading (NLP3). SEM confirmed spherical nano-vesicles, while FTIR, DSC, and XRD analyses demonstrated successful incorporation of lutein into the lipid bilayer and its transformation from a crystalline to an amorphous state. Under simulated gastrointestinal conditions, NLP3 showed the highest lutein release (≈ 73.0%) and micellar bioaccessibility (45.2%), representing approximately a two-fold enhancement compared with lower-loading formulations. Storage studies revealed superior lutein retention in NLP3 (≈ 85%, 78%, and 75% after 1, 15, and 30 days, respectively). Moreover, DPPH assays indicated that nanoliposomal encapsulation effectively preserved lutein antioxidant activity during storage and enabled sustained radical scavenging behavior. Overall, this work demonstrates an integrated valorization strategy combining green extraction and nanoliposomal delivery to enhance the stability, release, and bioaccessibility of lutein from pumpkin agro-industrial waste, highlighting the potential for developing sustainable, value-added functional food ingredients, nutraceuticals, and food fortification systems.

Introduction

Lutein-rich agro-industrial by-products, such as pumpkin peels and seeds, represent an underutilized resource that can be transformed into high-value functional ingredients, aligning with global priorities in sustainable food production and circular bioeconomy¹. Valorizing these residues through the recovery of carotenoids not only mitigates environmental burdens associated with waste disposal but also generates economic value by supplying natural colorants and bioactive compounds for food, nutraceutical, and pharmaceutical applications^2–5.

Over the past few years, several studies have focused on extracting carotenoids from pumpkin and other plant by-products using greener techniques such as supercritical CO₂ and alternative solvent systems¹, demonstrating that these matrices can yield lutein-rich fractions suitable for food and nutraceutical applications^6,7. Supercritical CO2 extraction has been particularly recognized as a green, food-grade technology capable of efficiently recovering oxidation-sensitive lipophilic bioactives while preserving their functional quality, with recent studies providing quantitative evidence on process optimization and quality retention under supercritical conditions^8,9.

In parallel, nanoscale delivery systems, particularly liposomes, nanoliposomes (NLPs), nanoemulsions, and biopolymer-based particles, have been widely investigated for improving the dispersibility, stability, and gastrointestinal performance of lutein, with recent reports showing enhanced micellarization and cellular uptake compared with non-encapsulated lutein^4,10,11. However, most existing works either optimize extraction from agro-waste¹² or evaluate nanoencapsulation of purified lutein^13,14, and only a limited number integrate green extraction of lutein from specific waste streams, such as pumpkin processing residues^15–17, with subsequent NLP formulation and in vitro digestion-based assessment of bioaccessibility under physiologically relevant conditions. Although these studies demonstrate the feasibility of carotenoid recovery or lutein encapsulation separately, limited attention has been paid to the integrated impact of green extraction combined with nanoliposomal delivery on lutein’s physical state and gastrointestinal performance. This limitation mainly arises from lutein’s crystalline structure, hydrophobicity, and susceptibility to oxidative degradation, which restrict its dispersion and micellar incorporation during digestion⁶. Compared with conventional lutein sources such as marigold, pumpkin peel offers advantages including wide availability, low cost, and compatibility with circular bioeconomy frameworks¹⁵.

Consequently, there remains a notable research gap regarding how encapsulating lutein extracted from pumpkin agro-industrial by-products into food-grade NLPs influences its structural state, release behavior, and gastrointestinal bioaccessibility compared with non-encapsulated counterparts. Addressing this gap is essential for rational design of sustainable, value-added food ingredients derived from plant waste and for translating nanoencapsulation strategies into practical formulations compatible with industrial processing.

To the best of our knowledge, this is among the first studies to combine supercritical CO₂ extraction of lutein from pumpkin peel with nanoliposomal encapsulation and systematic evaluation of structural state, release behavior, and in vitro bioaccessibility. The present study therefore aimed to (i) extract a lutein-rich carotenoid fraction from pumpkin processing waste, (ii) encapsulate this extract into NLPs and characterize their physicochemical, morphological, and structural properties, and (iii) evaluate lutein release and in vitro gastrointestinal bioaccessibility from the NLP system, with the overarching objective of demonstrating an integrated valorization pathway that enhances the functional value of lutein derived from agro-industrial residues.

Materials and methods

Raw materials and chemicals

Fresh Mammoth Gold pumpkins (Cucurbita maxima) were purchased from a local market in Mashhad, Iran (autumn 2024). Peels were washed, cleaned, dried in a vacuum oven (40 °C, 14 h), ground (Retsch ZM 200), sieved (60-mesh, < 250 μm), and stored at − 20 °C. Food-grade ethanol (Merck KGaA) was used for lipid phase preparation. Analytical solvents (n-hexane, acetone, methanol, acetonitrile, HPLC grade) and lutein standard (≥ 96%) were purchased from Sigma-Aldrich (USA) and Merck KGaA. Digestion reagents (pepsin ≥ 2500 U/mg, pancreatin ≥ 100 U/mg, bile salts) were obtained from Sigma-Aldrich and used without further purification.

Preparation of lutein-rich carotenoid extract

Lutein-rich carotenoid extract was prepared from pumpkin peel powder (Sect. “Raw materials and chemicals”) using a laboratory-scale supercritical CO₂ extractor (SFT-120XW, Supercritical Fluid Technologies Inc., Newark, DE, USA) equipped with a 10 mL stainless-steel extraction vessel. For each run, 0.40 g of peel powder was loaded into the extraction cell, and supercritical CO₂ extraction was performed for 40 min at a CO₂ flow rate of 1.5 mL/min under selected pressure (25–35 MPa) and temperature (40–70 °C) conditions to optimize carotenoid yield. The extracts were collected in amber vials, rinsed with n-hexane, concentrated under a gentle stream of nitrogen, re-dissolved in n-hexane, and stored at − 20 °C in the dark until HPLC analysis^18,19.

Preparation of lutein-loaded NLPs (Lutein-NLPs)

Lutein-loaded nanoliposomes (Lutein-NLPs) were prepared from pumpkin peel carotenoid extract using ethanol injection followed by probe ultrasonication. Lecithin (500 mg, Lipoid S100) and Tween 80 (50 mg) were dissolved in 10 mL ethanol (96%) by magnetic stirring (1200 rpm, 20 min, 55 °C) to form the lipid phase. Carotenoid extract equivalent to 5 mg lutein (lutein/lecithin ratio: 0.5, 0.75, 1.0, 1.5% w/w) was added and mixed thoroughly. This ethanolic lipid-extract solution was rapidly injected into 120 mL phosphate-buffered saline (PBS; 0.01 M, pH 7.4) at 55 °C under stirring (800 rpm). The crude liposomes were ethanol-free by rotary evaporation (40 °C, 100 mbar), then probe-sonicated (240 W, 30 min, 1 s on/1 s off) to obtain NLPs. Final Lutein-NLPs were nitrogen-flushed, stored at 4 °C in the dark, and analyzed within 48 h²⁰.

Quantification of lutein

Lutein was quantified using an Agilent 1100 HPLC system (Agilent Technologies, USA) equipped with a quaternary pump, vacuum degasser, autosampler, column oven, and UV-Vis detector. Separation was achieved on a C18 reversed-phase column (250 × 4.6 mm, 5 μm) at 30 °C. The mobile phase consisted of acetonitrile/methanol/water (80:15:5, v/v/v) at a flow rate of 1.0 mL/min using an isocratic elution program. Twenty microliters (20 µL) of samples and standards were injected, and lutein was detected using a Diode Array Detector (DAD) at a wavelength of 450 nm. Identification was based on a retention time of approximately 2.31 min and comparison of spectral profiles () with authentic standards. Quantification was performed using external calibration curves constructed from the lutein standard (R² > 0.999)²¹.

Determination of encapsulation efficiency and loading capacity

Encapsulation efficiency (EE) and loading capacity (LC) of lutein in NLPs were determined by quantifying non-encapsulated lutein from pumpkin peel carotenoid extract in the supernatant after separation from Lutein-NLPs²². Freshly prepared nanoliposomal dispersions were ultracentrifuged (25,000 × g, 1 h, 4 °C) to separate free lutein in the aqueous supernatant from the liposomal pellet. Lutein concentration in the supernatant was measured by HPLC (Sect. “Quantification of lutein”), and EE (%) and LC (%) were calculated using standard equations:

and

: the total mass of lutein initially added to the formulation.

the mass of lutein measured in the separated supernatant after centrifugation.

: the mass of lutein encapsulated in the NLPs.

is the total mass of lipid components.

Particle size, polydispersity index, zeta potential and stability

Particle size, polydispersity index (PDI), and zeta potential of the Lutein-NLPs were measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano series (Malvern Instruments Ltd., UK). Prior to analysis, samples were appropriately diluted with phosphate‑buffered saline to avoid multiple scattering, and measurements were carried out at 25 °C in disposable polystyrene (for size/PDI) and folded capillary (for zeta potential) cells. The intensity‑weighted mean hydrodynamic diameter (Z‑average) and PDI were recorded to describe the average size and distribution width of the NLPs, while zeta potential values were used to assess the surface charge and colloidal stability of the dispersions¹⁰.

Lutein stability in the nanoliposomal dispersions was also evaluated during storage (at days 0, 15 and 30) after centrifugation at 12,000 rpm for 20 min at 4 °C, and the percentage of retained lutein was calculated as:

where and are the lutein concentrations measured in the dispersion before and after centrifugation, respectively, as determined by HPLC or UV–Vis spectrophotometry.

Morphological analysis by SEM

Morphology of Lutein-NLPs was examined using a JEOL JSM-6390LV scanning electron microscope (SEM, JEOL Ltd., Tokyo, Japan). Freshly prepared dispersions were diluted 1:1000 in deionized water, and 10 µL droplets were placed on aluminum stubs with carbon conductive tape. Samples were air-dried in the dark at room temperature, then sputter-coated with gold (5 nm thickness) using a Quorum Q150R S sputter coater (Quorum Technologies, UK). Imaging was performed at 10 kV accelerating voltage with a working distance of 8–10 mm. SEM micrographs qualitatively assessed liposome shape, surface morphology, and aggregation, complementing DLS size/PDI data (Sect. “Particle size, polydispersity index, zeta potential and stability”)¹⁰.

Structural and thermal characterization (FTIR, DSC, XRD)

Structural and thermal properties of Lutein-NLPs were evaluated by Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and X-ray diffraction (XRD). FTIR spectra of pumpkin peel carotenoid extract, blank-NLP, and Lutein-NLPs were recorded (4000–400 cm⁻¹, 4 cm⁻¹ resolution) using a PerkinElmer Spectrum Two spectrometer to identify phospholipid-carotenoid interactions through characteristic peak shifts or intensity changes. DSC analysis of extract, lipids, physical mixtures, and Lutein-NLPs (~ 5 mg samples) was performed using a TA Instruments DSC Q200 (10 °C/min heating rate, 30–300 °C, sealed aluminum pans under nitrogen) to detect changes in melting transitions and enthalpy indicative of lutein amorphization upon encapsulation. XRD patterns of powdered samples were obtained using a Bruker D8 Advance diffractometer (2θ = 5–60°, Cu Kα radiation) to distinguish crystalline carotenoid peaks from amorphous lipid halos, confirming the physical state and dispersion of carotenoids within the nanoliposomal matrix²³.

Antioxidant activity assay

Antioxidant activity of pumpkin peel carotenoid extract, blank-NLP, and Lutein-NLPs was evaluated using the DPPH radical scavenging assay. Freshly prepared 0.15 mM DPPH in methanol (2 mL) was mixed with diluted samples (equivalent to 0.1–1.0.1.0 mg/mL extract) and incubated in the dark at room temperature for 30 min. Absorbance was measured at 517 nm using a Shimadzu UV-1800 spectrophotometer. Methanol served as blank and Trolox (0.01–0.1 mM) as positive control. DPPH scavenging activity (%) was calculated as:

where A₀ is the absorbance of the control and A_s is the absorbance of the sample. All measurements were performed in triplicate.

Static in vitro gastrointestinal digestion and assessment of lutein release and micellar bioaccessibility

A static INFOGEST in vitro gastrointestinal digestion model was employed to simulate gastric (2 h) and intestinal (2 h) phases at 37 °C under shaking conditions at 120 rpm. For the gastric phase, 5 mL of Lutein-NLPs dispersion was mixed with 5 mL simulated gastric fluid (SGF; containing NaCl, HCl to adjust pH to 2.5) and porcine pepsin (2000 U/mL). After gastric digestion, the pH was adjusted to 7.0 using 0.5 M NaOH. For the intestinal phase, 10 mL simulated intestinal fluid (SIF; containing NaCl, CaCl₂ to adjust pH to 7.0), bile salts (10 mM), porcine pancreatin (100 U/mL), and lipase (500 U/mL) were added, followed by incubation for 2 h²⁴.

Lutein release was determined by centrifugation (25,000 × g, 1 h, 4 °C) of post-digestion mixtures, hexane extraction of the supernatant, and spectrophotometric quantification at 445 nm (hexane blank). Micellar bioaccessibility was assessed by ultracentrifugation of post-intestinal digesta (100,000 × g, 20 min, 4 °C) to remove large particulates and undigested material. The micelle-rich supernatant was carefully collected and subjected to hexane extraction, followed by measurement at 445 nm. Hexane extraction efficiency was validated (> 90% recovery)²⁵.

where M_free is the amount of lutein recovered in the hexane phase after digestion, and M_encapsulated is the amount of lutein remaining associated with the liposomal fraction.

where M_micellar is the amount of lutein present in the micelle‑rich fraction and M_initial is the initial amount of lutein present in the digested sample before in vitro gastrointestinal digestion. This calculation follows the standard definition used in INFOGEST protocols for assessing the fraction of bioactive compounds solubilized in mixed micelles available for intestinal uptake²⁶.

Statistical analysis

Statistical analyses were performed using SPSS software (IBM SPSS Statistics, Version 26.0, IBM Corp., Armonk, NY, USA). All experiments were conducted at least in triplicate and data were expressed as mean ± standard deviation. One‑way analysis of variance (ANOVA) was applied to evaluate differences among treatments, and when significant effects were detected, Tukey’s post hoc test was used for multiple comparisons, with a significance level set at p < 0.05.

Results and discussion

Lutein content and chromatographic characteristics of the carotenoid extract

The HPLC chromatogram of the lutein standard showed a single dominant peak with a retention time of approximately 2.31 min and a λmax​ of 446 nm (Fig. 1a), confirming the specificity of the analytical method. In contrast, the chromatogram of the supercritical CO₂ extract obtained from Mammoth Gold pumpkin peel exhibited a major peak at around 3.99 min (Fig. 1b) with a of 446 nm, identical to the standard. The observed retention time shift (2.31 min → 3.99 min) between pure standard and peel extract is attributable to two main factors: (i) solvent mismatch, as the standard was dissolved in methanol while the extract was redissolved in n-hexane, and (ii) matrix effects from co-extracted compounds. This solvent-induced shift is consistent with observations by Pothinam et al.⁸ in supercritical extraction studies of lipophilic compounds. Peak identification was rigorously confirmed by matching retention time, peak area response ratios, and UV-Vis spectral scans (). Furthermore, the spectral purity index at the peak apex was calculated to be > 0.99, indicating no significant co-elution with other matrix components. The negative peak observed in Fig. 1b is attributed to a solvent front disturbance or refractive index mismatch caused by the injection of the n-hexane dissolved extract into the aqueous-organic mobile phase, a common occurrence in complex matrix analysis^21,27,28.

Fig. 1.

HPLC chromatograms of pure lutein (a) and lutein extracted from pumpkin peel using supercritical CO₂ (b).

Quantitative analysis revealed that the supercritical CO₂ extraction yielded a carotenoid-rich extract with a lutein concentration of 12.3 ± 0.6 mg/g of dried peel powder. Based on the mass of the extract obtained from 0.40 g of peel powder, the extraction yield was calculated to be 2.8 ± 0.2% w/w. This yield is competitive with the 2.5–3.0% range reported by Stajčić et al.¹⁷ for pumpkin waste extraction, and higher than the 1.5% yield often observed in solvent-based extractions of pumpkin peel¹⁸. These quantitative metrics confirm the efficiency of the optimized extraction conditions in recovering bioactive lutein from pumpkin by-products.

From a broader perspective, the predominance of lutein in the supercritical CO₂ extract aligns with recent studies showing that pumpkin by‑products, including peel and pulp, are particularly rich in lutein relative to other carotenoids^1,29,30. Supercritical CO₂ has been demonstrated as an efficient and selective solvent for carotenoids, enabling high lutein recoveries from pumpkin and other agro‑industrial residues while minimizing thermal and oxidative degradation compared with conventional solvent extraction^20,30,31. These findings indicate that Mammoth Gold pumpkin peel can be effectively valorized as a sustainable source of lutein‑rich carotenoid extract suitable for further nanoencapsulation and incorporation into functional food formulations.

Characteristics of lutein-NLPs

The physicochemical properties, in vitro release, gastrointestinal bioaccessibility, and storage stability of lutein-NLPs (NLP1–NLP4) are summarized in Table 1. EE increased significantly with rising lutein/lecithin ratio, reaching a maximum in NLP3 (86.3%), followed by a slight decline in NLP4 (79.9%). This trend mirrors findings by Zhao et al.³², where an optimal lipid ratio maximized encapsulation efficiency before saturation occurred. This behavior suggests that intermediate lutein loading promotes optimal molecular accommodation within the phospholipid bilayer, whereas excessive lutein may induce partial aggregation or saturation of lipid domains, thereby reducing encapsulation capacity. Similar trends have been reported for lutein and other carotenoids encapsulated in lipid-based nanocarriers, where balanced bioactive/lipid ratios govern membrane organization and loading efficiency^32,33.

Table 1.

Physicochemical properties, release, bioaccessibility, and stability of lutein‑loaded NLPs (mean ± SD).

Formulation	EE (%)	Z average (nm)	PDI	Zeta potential (mV)	Lutein release (%)	Lutein bioaccessibility (%)	Stability day 1 (%)	Stability day 15 (%)	Stability day 30 (%)
NLP1	65.11 ± 0.22 d	272.07 ± 4.21 a	0.44 ± 0.11 b	−24.53 ± 0.07 b	35.01 ± 0.22 d	31.1 ± 2.2 d	80.2 ± 1.1	64.3 ± 2.4	50.5 ± 3.0
NLP2	74.41 ± 0.12 c	203.77 ± 2.11 c	0.34 ± 0.15 c	−26.61 ± 0.37 c	44.11 ± 0.12 c	34.1 ± 1.2 c	84.6 ± 1.0	61.5 ± 2.6	60.2 ± 2.8
NLP3	86.32 ± 0.20 a	109.63 ± 1.30 e	0.18 ± 0.12 e	−22.14 ± 0.23 a	73.02 ± 0.20 a	45.2 ± 2.0 a	85.1 ± 0.9	78.0 ± 2.3	75.4 ± 2.5
NLP4	79.85 ± 0.21 b	213.54 ± 2.15 b	0.26 ± 0.11 d	−29.12 ± 0.24 e	59.05 ± 0.21 b	40.5 ± 2.1 b	81.3 ± 1.2	70.4 ± 2.7	66.1 ± 2.9
Blank-NLP	64.11 ± 0.22 d	170.8 ± 3.17 d	0.54 ± 0.15 a	−28.44 ± 0.57 d	N/A	N/A	N/A	N/A	N/A

Values labeled with different lowercase letters within each column indicate statistically significant differences (p < 0.05). ND: Not detected.

NLP1–NLP4 represent lutein-loaded nanoliposome formulations prepared with increasing lutein/lecithin ratios: NLP1 (0.5), NLP2 (0.75), NLP3 (1.0), and NLP4 (1.5) % w/w. Blank-NLP refers to nanoliposomes without lutein; therefore, encapsulation efficiency, release, bioaccessibility, and stability parameters were not applicable and are indicated by N/A.

Particle sizes ranged from approximately 109 to 272 nm, with NLP3 exhibiting the smallest mean diameter and lowest PDI, indicating a more homogeneous dispersion. This size range (109–272 nm) falls within the optimal nanoscale domain (100–300 nm) described by Liu et al.³⁴ for efficient intestinal uptake of lipids. All formulations displayed negative zeta potentials (− 22.1 to − 29.1 mV), reflecting electrostatic stabilization of vesicles and favorable colloidal stability. These values are comparable to the − 20 to − 35 mV range reported for lecithin-based lutein carriers by Algan et al.⁴, confirming sufficient charge repulsion to prevent aggregation. Reduced particle size and narrow size distribution are known to enhance digestive accessibility and lipid hydrolysis, thereby improving release and intestinal uptake of lipophilic compounds^35,36.

Lutein release and micellar bioaccessibility were significantly higher in NLP3 and NLP4 than in NLP1 and NLP2, with NLP3 achieving the highest release (73.0%) and bioaccessibility (45.2%). This enhanced gastrointestinal performance can be attributed to the amorphous dispersion of lutein within the lipid matrix (confirmed by DSC/XRD), combined with smaller particle size, which collectively facilitate interaction with bile salts and digestive enzymes. Comparable improvements in carotenoid bioaccessibility following nanoliposomal encapsulation have been reported previously, emphasizing the role of molecular dispersion and nanoscale lipid organization in promoting mixed micelle formation^33,37.

During storage, lutein retention decreased gradually in all formulations; however, NLP3 consistently exhibited superior stability (≈ 85%, 78%, and 75% at days 1, 15, and 30, respectively), whereas NLP1 showed the greatest losses. The improved stability of NLP3 likely arises from effective embedding of lutein within the bilayer, which provides physical shielding against oxygen and light while minimizing recrystallization. Similar protective effects of nanoliposomes on carotenoid stability have been documented in food-grade lipid delivery systems^32,35.

Overall, NLP3 (1.0% lutein/lecithin) offered the most balanced performance in terms of EE, particle homogeneity, release behavior, gastrointestinal bioaccessibility, and storage stability. These findings highlight the critical importance of optimizing lutein/lipid ratios to achieve favorable membrane structure and functional performance, supporting the suitability of nanoliposomal systems as efficient carriers for enhancing lutein stability and intestinal availability.

Morphological features of nano-liposomes (SEM)

The morphology of lutein-loaded nanoliposomes (Lutein-NLP) was examined by SEM to determine vesicle shape, surface characteristics, and aggregation state (Fig. 2). All formulations exhibited predominantly spherical to quasi-spherical vesicles with relatively smooth surfaces, confirming successful formation of nanoscale liposomal structures. NLP3 demonstrated the most uniform morphology with minimal aggregation, whereas other formulations showed slight clustering, likely resulting from partial dehydration during SEM sample preparation. This spherical morphology is characteristic of liposomes prepared via ethanol injection, as described by Wang et al.²², and confirms the absence of irregular precipitation.

Fig. 2.

SEM micrographs of blank and lutein-loaded nanoliposomes.

The particle dimensions observed by SEM were consistent with the hydrodynamic diameters obtained via DLS, corroborating the formation of well-defined nanoliposomes. The superior morphological homogeneity of NLP3, as reflected in its lower PDI, indicates a more uniform lipid bilayer organization. Similar findings have been reported in recent studies where optimized lipid ratios minimized structural heterogeneity in nanoliposome systems^10,22.

From a functional standpoint, well-defined spherical morphology and narrow size distribution are critical parameters influencing colloidal stability, digestive interactions, and cellular uptake. Smaller, uniformly dispersed nano-vesicles provide a larger effective surface area and improved accessibility to digestive enzymes and bile salts, thereby facilitating enhanced release and micellar incorporation of lipophilic bioactives^4,38. Recent reviews on food nanoliposomes have also emphasized that morphological uniformity reduces vesicle fusion and aggregation, leading to improved gastrointestinal performance and bioaccessibility^4,35.

Mechanistically, the balanced lutein/lecithin ratio in NLP3 likely promotes optimal lipid packing, reducing defects in the bilayer and stabilizing nano-vesicle formation. Elevated lutein loading (e.g., in NLP4) can perturb membrane regularity due to steric crowding, resulting in increased heterogeneity and occasional clustering. Comparable behavior has been documented in studies of carotenoid-loaded liposomes, where excessive bioactive loading impaired membrane cohesion and increased PDI values^33,36.

Overall, SEM results support the physicochemical characterization and functional performance of the nanoliposomes. The structural uniformity and nanoscale morphology observed, particularly in NLP3, align with enhanced colloidal stability and improved digestive functionality, reinforcing the role of optimized morphological features in effective nanoencapsulation of hydrophobic bioactives.

FTIR spectra and molecular interactions

FTIR spectroscopy was employed to investigate molecular interactions between lutein and the NLP matrix and to confirm successful encapsulation (Fig. 3). The characteristic absorption bands for pure lutein, blank NLP, and Lutein-NLP are summarized in Table 2. Pure lutein exhibited distinctive peaks associated with its conjugated polyene chain (C–H stretching at 3015 cm⁻¹), conjugated carbonyl groups (1814 cm⁻¹), and C–O stretching (1263 cm⁻¹). Blank NLP showed a broad O–H band at 3587 cm⁻¹ and a strong ester carbonyl (C = O) peak at 1758 cm⁻¹. Upon encapsulation, the Lutein-NLP spectrum revealed minor but significant shifts: the C–H band shifted from 3015 to 3024 cm⁻¹, the ester carbonyl shifted from 1758 to 1780 cm⁻¹, and the C–O band shifted from 1263 to 1276 cm⁻¹. These spectral perturbations are consistent with findings by Liu et al.³⁴ and Shavronskaya et al.³⁹, indicating the formation of weak intermolecular interactions, such as hydrogen bonding between lutein hydroxyls and lipid ester groups, as well as hydrophobic interactions within the bilayer. Notably, the absence of new peaks and the preservation of fundamental functional groups confirm that lutein was physically entrapped rather than chemically altered. This physical entrapment is crucial for maintaining the chemical integrity of the bioactive compound, a key requirement for food-grade applications³⁹. The reduced intensity of some lutein peaks in the Lutein-NLP spectrum further supports the deep incorporation of lutein into the lipid matrix. Overall, FTIR analysis validates the successful encapsulation and suggests that the intermolecular forces formed contribute to the structural stability of the nanoliposomal system.

Fig. 3.

FTIR spectra showing molecular interactions between lutein and lipid bilayers in blank and lutein-loaded nanoliposomes.

Table 2.

Characteristic FTIR absorption bands (cm⁻¹) of Pure Lutein, Blank-NLP, and Lutein-NLP.

Functional Group	Pure Lutein (cm⁻¹)	Blank-NLP (cm⁻¹)	Lutein-NLP (cm⁻¹)
O–H Stretching (Hydrogen bonding)	-	3587	3587
C–H Stretching (Aliphatic)	3015	-	3024
C = O Stretching (Ester/Carotenoid)	-	1758	1780
Conjugated C = C stretching	1814	-	1896
C–O Stretching (Fingerprint)	1263	-	1263

Thermal behavior by DSC

DSC thermograms of pure lutein, Blank-NLP, and lutein-loaded nanoliposomes are presented in Fig. 4. Pure lutein exhibited a characteristic thermal event associated with its melting point around 244 °C, confirming its crystalline nature. The Blank-NLP thermogram showed a broad endothermic event at approximately 233 °C; however, considering that lecithin phase transitions typically occur below 100 °C, this event is attributed to the thermal decomposition of the phospholipid components rather than a bilayer phase transition.

Fig. 4.

DSC thermograms showing the thermal behavior of pure lutein, blank nanoliposomes (Blank-NLP), and lutein-loaded nanoliposomes.

Crucially, the characteristic thermal signature of lutein at 244 °C was absent in the thermogram of the Lutein-NLP. Instead, a broad thermal transition was observed at approximately 125 °C, indicating the loss of long-range crystalline order. This disappearance of the melting peak confirms the amorphous state, a phenomenon consistently reported for encapsulated carotenoids by Liu et al.³⁴. While the thermograms exhibit a baseline drift typical of high-temperature decomposition in lipid-carotenoid systems, the distinct disappearance of the crystalline lutein signal and the shift in thermal behavior toward lower temperatures strongly support the transformation of lutein from a crystalline to an amorphous state within the lipid matrix. The presence of this transition at 125 °C suggests strong molecular interactions between lutein and the lipid bilayer, likely involving hydrogen bonding and hydrophobic effects that disrupt crystal lattice formation.

The amorphous physical state of the encapsulated lutein is functionally advantageous, as it enhances solubilization and release efficiency compared to crystalline forms, which resist solubilization. These findings align with previous reports where encapsulation-induced amorphization was linked to enhanced stability and bioaccessibility of lipophilic bioactives³⁴.

Crystallinity and physical state by XRD

X-ray diffraction patterns of pure lutein, Blank-NLP, and lutein-NLPs are presented in Fig. 5. Pure lutein exhibited several sharp and intense diffraction peaks at 2θ ≈ 11.08°, 19.44°, 20.62°, 30.06°, 44.22°, and 56.02°, confirming its highly crystalline nature. In contrast, Blank-NLP showed broader and less intense reflections at 2θ ≈ 11.08°, 14.32°, 22.96°, 29.44°, 30.52°, 44.59°, and 56.44°, characteristic of semi-crystalline phospholipid structures.

Fig. 5.

XRD diffractograms of lutein, Blank-NLP, and Lutein-NLP.

Notably, the characteristic crystalline peaks of lutein completely disappeared in the diffraction pattern of lutein-loaded nanoliposomes, which instead displayed broad halos in the range of 2θ ≈ 17–26°. This loss of crystallinity matches the XRD patterns of amorphized carotenoids described by Ahmadzadeh et al.¹¹. This behavior indicates successful incorporation of lutein into the lipid bilayer and its transformation from a crystalline to an amorphous state within the nanoliposomal matrix. The reduced peak intensity and peak broadening further reflect disruption of long-range molecular order and formation of a less organized lipid structure⁴⁰-⁴¹.

These XRD findings are consistent with DSC results and confirm that lutein was molecularly dispersed within the lipid phase rather than deposited on the particle surface. The amorphous physical state of encapsulated lutein is advantageous for enhancing loading efficiency, reducing recrystallization during storage, and promoting sustained release and improved bioaccessibility¹³.

Antioxidant activity of lutein-loaded nano-liposomes

Antioxidant activity profiling was performed on both free lutein and encapsulated lutein (Lutein-NLP) over a 15-day storage period to assess the stability and bioaccessibility profile. As shown in Fig. 6, free lutein exhibited high initial radical scavenging activity (approx. 67% at Day 0), which is consistent with the direct and unimpeded interaction of the molecule with DPPH radicals. However, this activity decreased sharply to approximately 45% by Day 15, confirming the known susceptibility of unencapsulated lutein to rapid oxidation and degradation under storage conditions. This degradation rate is comparable to the rapid loss of activity in free carotenoids reported by García-Morales et al.⁴². In contrast, Lutein-NLP displayed significantly lower initial activity (≈ 15% at Day 0). This lower initial value is attributed to the physical barrier effect imposed by the nanoliposomal membrane, which limits the immediate accessibility of the encapsulated lutein to the external DPPH solution. Crucially, the activity of Lutein-NLP demonstrated a controlled, gradual increase over the storage period, reaching ≈ 28% by Day 15. This sustained release profile is consistent with the controlled antioxidant activity observed in lipid nanocarriers by Karim et al.⁴³.

Fig. 6.

Antioxidant activity (DPPH radical scavenging) of free lutein, lutein-loaded nanoliposomes (Lutein-NLP), and blank nanoliposomes (Blank-NLP) during storage.

This upward trend does not indicate an increase in inherent antioxidant potency, but rather may reflect the time-dependent reorientation and increased accessibility of bioactive lutein molecules from the stable lipid matrix into the assay medium. This pattern confirms that the nanoliposomal formulation provides effective protection against initial degradation while ensuring a sustained and functional availability of lutein over time, a hallmark of advanced controlled-release systems.

Nanoencapsulation has been widely reported to enhance antioxidant stability and extend functional activity by protecting labile bioactives from oxidative stress and degradation⁴⁴. Nanoliposomes can shield encapsulated compounds from environmental oxidants and control release, leading to prolonged radical scavenging activity over the storage period⁴³. Nanoliposome carriers have been shown to preserve the antioxidant capacity of encapsulated phytochemicals such as carotenoids and vitamins better than their free forms during storage (e.g., β-carotene and phenolic antioxidants), highlighting enhanced retention and sustained activity⁴⁵.

The observed increase in DPPH activity for Lutein-NLP over time suggests controlled release of lutein from the lipid bilayer, whereby lutein gradually becomes available to interact with the DPPH radical. Sustained release behavior from lipid nanocarriers has been reported in recent studies, where nanoliposomal encapsulation delayed degradation and improved antioxidant persistence compared with free molecules⁴².

Mechanistically, the protective effect of nanoliposomes can be attributed to the bilayer matrix providing a physical barrier to oxygen and free radicals, reducing premature lutein oxidation and enabling continuous accessibility of active lutein molecules. This aligns with recent literature suggesting that nanoliposome formulations maintain antioxidant efficacy during storage and digestion by limiting direct exposure of sensitive compounds to adverse conditions^39,46.

Overall, the sustained antioxidant performance of Lutein-NLP, even in later storage days, indicates that nanoliposomal encapsulation improves both storage stability and functional longevity of lutein’s radical-scavenging activity, reinforcing the advantage of nanocarrier systems for delivering sensitive lipophilic antioxidants in food and nutraceutical applications. It should be noted that while the DPPH assay is a standard and reliable method for evaluating radical scavenging activity in carotenoid systems, employing additional assays such as ABTS or FRAP in future studies could provide a more comprehensive antioxidant profile.

In vitro release profile of lutein

The in vitro release behavior of lutein from nanoliposomal formulations was evaluated under simulated gastrointestinal conditions (Table 1). Lutein release increased significantly with increasing lutein/lecithin ratio, with NLP1 and NLP2 showing lower release (≈ 35.0% and ≈ 44.1%, respectively) and NLP3 achieving the highest release (≈ 73.0%). This release efficiency is higher than the 50–60% release reported for polymeric nanocapsules by Han et al.¹⁴, demonstrating the superior permeability of lipid bilayers. A slight decline was observed in NLP4 (≈ 59.0%), suggesting that excessive lutein loading may hinder optimal release.

The release patterns observed here are consistent with findings in recent studies where nanocarrier systems demonstrated enhanced release of encapsulated lipophilic compounds under simulated digestive conditions. Intermediate bioactive loading often provides a more favorable lipid packing arrangement within the bilayer, resulting in improved access to digestive enzymes and bile salts, which facilitate micellar solubilization and compound efflux. This is supported by studies showing that optimal lipid: bioactive ratios maximize release efficiency, whereas overly high loading may cause aggregation or tighter packing that restricts molecular mobility and slows release⁴⁷.

Moreover, the observed release behavior correlates well with the DSC and XRD data: NLP3 exhibited the greatest amorphous character and smallest particle size, which are key factors that enhance accessibility of digestive lipases and mixed micelle formation during intestinal digestion. Nanostructured lipid carriers with reduced crystallinity and increased surface area have been demonstrated to promote faster and more sustained release profiles in simulated gastrointestinal models by enabling easier interaction with enzymatic systems²⁴.

Mechanistically, release from lipid-based carriers like nanoliposomes depends on multiple interrelated factors: the integrity and fluidity of the lipid bilayer, the degree of bioactive dispersion, and the digestive environment created by bile salts and pancreatic enzymes. When bilayer packing is optimal (as in NLP3), enzyme access and lipid digestion are enhanced, facilitating micelle formation and release of lutein into the aqueous phase. Excessive lutein loading (e.g., in NLP4) may induce localized packing defects or microdomain aggregation that slow the desorption process, as has been discussed in other nanolipid systems encapsulating hydrophobic nutrients^14,28.

Overall, the in vitro release data indicate that NLP3 provides the most favorable balance of bilayer organization and release kinetics, supporting its superior gastrointestinal performance and explaining its enhanced micellar bioaccessibility. These findings align with recent literature emphasizing the importance of carrier architecture and physical state in governing release behavior of lipophilic nutraceuticals^24,38.

Micellarization and bioaccessibility of lutein under simulated gastrointestinal conditions

The micellarization efficiency and gastrointestinal bioaccessibility of lutein from the nanoliposomal formulations were evaluated using a static in vitro digestion model (Table 1). Lutein bioaccessibility increased with rising lutein loading, reaching the highest values in NLP3 (45.2%) and NLP4 (40.5%), which were approximately two-fold higher than those of NLP1 and NLP2. Notably, the bioaccessibility of NLP3 was significantly higher than that of non-encapsulated free lutein, which typically ranges between 15 and 25% in solid food matrices under similar digestion conditions due to limited micellarization^10,32. This represents an approximately two-fold enhancement compared to the free form. Furthermore, this value aligns well with recent literature on optimized lutein nanoliposomes (30–50%)^23,47, confirming the efficacy of the supercritical CO₂ extract encapsulated in NLPs. This enhancement reflects improved incorporation of lutein into mixed micelles during the intestinal phase.

Bioaccessibility is defined as the fraction of a compound solubilized into mixed micelles during digestion and thus available for intestinal uptake. Efficient micellar incorporation is a critical step for lipophilic nutrients like carotenoids, where micelle formation depends on lipid digestion products such as free fatty acids and monoacylglycerols produced in the intestinal phase. Nanocarrier systems can significantly enhance this process by facilitating triglyceride hydrolysis and increasing the surface area available for bile salt interaction, which together promote mixed micelle formation and carotenoid solubilization²⁵.

The superior bioaccessibility observed for NLP3 is consistent with its optimized release behavior, smaller particle size, and amorphous physical state, all of which collectively facilitate lipid digestion and micelle formation. Carotenoids embedded in more amorphous lipid matrices are more easily released into the aqueous phase and incorporated into mixed micelles compared to their crystalline counterparts, which tend to remain associated with solid clusters and resist incorporation²⁴.

In contrast, the marginally lower bioaccessibility of NLP4, despite its higher lutein content, suggests that excessive loading can promote tighter lipid packing or partial aggregation, potentially limiting accessibility of bile salts and digestive enzymes to the encapsulated lutein and thus reducing its micellarization efficiency. This phenomenon aligns with reports showing that an optimal lipid-bioactive ratio is essential for maximizing bioaccessibility, whereas overwhelming the carrier can hinder micelle formation dynamics⁴⁸.

Overall, NLP3 provided the most efficient micellarization and intestinal availability of lutein, highlighting the importance of balanced lutein/lipid ratios for maximizing gastrointestinal uptake. These results demonstrate that nanoliposomal encapsulation effectively enhances lutein bioaccessibility by promoting molecular dispersion and improved incorporation into digestive mixed micelles, which is consistent with current understanding of lipid digestion and carotenoid micellization mechanisms⁴⁹.

Proposed mechanisms for enhanced stability and bioaccessibility

Based on the combined physicochemical, structural, and gastrointestinal results, a preliminary mechanistic framework is proposed to explain the enhanced stability and bioaccessibility of lutein encapsulated in nanoliposomes.

Supercritical CO₂ extraction preserved lutein integrity with minimal thermal degradation prior to encapsulation. Subsequent nanoliposomal encapsulation facilitated molecular dispersion of lutein within the phospholipid bilayer, as indicated by DSC (loss of crystalline melting peak) and XRD (disappearance of characteristic crystalline peaks). Such transformation from crystalline to amorphous states has been associated with increased solubilization and release efficiency of carotenoids, as dispersed hydrophobic molecules interact more readily with digestive components than crystalline aggregates that resist solubilization^23,40.

Amorphous dispersion, together with reduced particle size and narrow size distribution, enhances interaction with digestive enzymes and bile salts during the intestinal phase⁴⁰. Efficient lipid digestion generates mixed micelles that facilitate solubilization and uptake of lipophilic bioactives; nanocarriers that provide larger surface area and accessible lipid interfaces promote micellar formation and bioaccessibility. This mechanism has been reported in recent investigations of lipid nanocarriers, where improved bioaccessibility correlates with enhanced enzymatic accessibility and micelle incorporation^24,25.

Embedding lutein within the lipid matrix also provides physical shielding against oxidative and environmental stress, contributing to superior storage stability and sustained antioxidant activity over time. Nanoliposomes can act as protective vehicles that limit exposure of sensitive compounds to oxygen and thermal stress while controlling release²⁶, consistent with findings that nanoliposome encapsulation enhances both stability and functional longevity of antioxidant bioactives⁵⁰.

The optimized formulation (NLP3) achieved a balanced lutein/lipid ratio that maximized membrane organization without inducing aggregation or steric hindrance. Excessive lutein loading can create packing strain within bilayers, reducing enzyme accessibility and micellar solubilization efficiency, similar to observations in other lipophilic nutrient delivery systems. Collectively, these results demonstrate that nanoliposomal delivery enhances lutein performance through synergistic effects of amorphization, nanoscale dispersion, and lipid-mediated protection, aligning with the current mechanistic understanding of lipid digestion and nutrient absorption pathways.

Conclusion

This study demonstrated an integrated and sustainable approach for valorizing pumpkin peel as a lutein-rich agro-industrial by-product through supercritical CO₂ extraction followed by nanoliposomal encapsulation. Lutein-loaded nanoliposomes exhibited favorable physicochemical characteristics, including nanoscale particle size, negative surface charge, and high EE, with the optimized formulation (NLP3, 1.0% lutein/lecithin) providing the best overall performance.

Structural analyses (SEM, FTIR, DSC, and XRD) confirmed successful incorporation of lutein into the lipid bilayer and its transformation from a crystalline to an amorphous state, which contributed to enhanced stability and gastrointestinal functionality. Under simulated digestion conditions, NLP3 achieved the highest lutein release (73.0%) and micellar bioaccessibility (45.2%), while also showing superior storage stability and sustained antioxidant activity compared with free lutein.

These findings highlight the critical role of balanced lutein/lipid ratios in governing membrane organization, molecular dispersion, and digestive behavior. Overall, nanoliposomal delivery effectively improved lutein stability, controlled release, and intestinal availability, demonstrating strong potential for developing value-added functional ingredients from pumpkin processing waste. This strategy offers a promising platform for incorporating lutein into food and nutraceutical formulations while supporting circular bioeconomy principles through agro-waste valorization.

Despite the promising results, this study has certain limitations. The in vitro gastrointestinal digestion model employed (INFOGEST) is a static system that does not fully replicate the dynamic physiological conditions of the human gut, such as peristalsis and varying pH gradients. Consequently, the bioaccessibility values reported here should be validated through in vivo studies in future work to confirm actual bioavailability. Additionally, the storage stability assessment was limited to 30 days; long-term stability studies under diverse environmental conditions would provide further insight into the shelf-life of the formulations. Furthermore, while the extract was successfully encapsulated, its application in real food matrices (e.g., beverages, dairy products, or bakery items) remains to be investigated. Future research will focus on scaling up the supercritical CO₂ extraction process for industrial feasibility and evaluating the safety and efficacy of pumpkin peel-derived nanoliposomal lutein in clinical or animal models.

These findings provide a scalable platform for converting pumpkin processing waste into high-value functional ingredients.

Author contributions

Mahya Sheykhzade: Conceptualization, Investigation, Methodology, Formal analysis, Writing - original draft; Vahid Hakimzade: Supervision, Project administration, Funding acquisition, Writing - review & editing; Esmaeel Ataye Salehi: Validation, Data curation, Visualization; Sharare Mohseni: Resources, Software, Formal analysis.

Data availability

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Declarations

Competing interests

The authors declare no competing interests.