Abstract
Propolis is a chemically complex natural product with recognized antioxidant potential, but its compositional heterogeneity and poor aqueous solubility complicate formulation and interpretation of in vitro release behavior. In this study, a nanostructured lipid carrier (NLC) based on Gelucire® 44/14 was developed as a physicochemical platform to modulate the accessibility of a selected Chilean ethanolic propolis extract. Propolis extracts from different origins were first screened using complementary antioxidant assays (DPPH, ABTS, ORAC, FRAP), leading to the selection of the Peñaflor extract, which exhibited the highest phenolic content (~41 mg GAE/g) and antioxidant capacity. The selected extract was incorporated into NLCs with encapsulation efficiencies above 90%, a narrow size distribution (~200 nm), and high stability over 90 days. Under simple aqueous conditions, propolis release remained limited (<15% over 6 h), consistent with diffusion- and partition-controlled transport. In simulated gastrointestinal media containing bile components, pronounced pH- and composition-dependent effects were observed. While fed-state intestinal conditions induced extensive morphological remodeling without increasing the analytically accessible fraction (<3% at 4 h), fasted-state intestinal media promoted a higher accessible fraction (~14% within 1 h) without complete carrier disruption, as confirmed by transmission electron microscopy. Preliminary cytocompatibility studies in HepG2 cells showed acceptable viability at 10–40 µg/mL and concentration-dependent effects at higher doses. Overall, this work demonstrates that bile components modulate propolis accessibility through dynamic partitioning and colloidal reorganization rather than simple carrier breakdown, providing a physicochemical framework for future digestion and absorption studies.
Introduction
Propolis is a chemically complex resinous material produced by bees through the transformation of plant-derived exudates, and it is widely recognized for its rich content of polyphenols, flavonoids, and other redox-active constituents [1]. Owing to this composition, propolis has been associated with antioxidant, antimicrobial, and anti-inflammatory properties, motivating sustained interest in pharmaceutical and nutraceutical research [2,3]. However, propolis does not represent a single, well-defined chemical entity; its composition is highly heterogeneous and strongly dependent on botanical source, geographic origin, and local environmental conditions, leading to pronounced variability even among samples collected within geographically close regions [4,5]. This intrinsic heterogeneity poses challenges for formulation development and complicates the interpretation of in vitro performance data.
From a physicochemical standpoint, additional limitations arise from the poor aqueous solubility and marked lipophilicity of many propolis constituents [6]. Under gastrointestinal conditions, these properties may restrict the fraction of compounds that become analytically accessible, resulting in low apparent release or activity values that reflect formulation-dependent partitioning phenomena rather than insufficient intrinsic chemical potential [7,8]. Consequently, conventional in vitro assays may underestimate or misrepresent the functional behavior of propolis if the formulation context, phase behavior, and analytical readouts are not carefully considered.
Several delivery systems have been explored to improve the stability and bioavailability of propolis and other polyphenol-rich natural extracts, including liposomes, polymeric particles, and solid lipid nanoparticles (SLNs) [9]. Liposomal systems are attractive due to their biocompatibility and ability to encapsulate both hydrophilic and lipophilic compounds [6]; however, their phospholipid bilayers may be destabilized under gastrointestinal conditions, particularly in the presence of bile salts and digestive surfactants, leading to membrane disruption and premature compound leakage [10]. Polymeric carriers such as PLGA-based microspheres can provide controlled release and protection against degradation, but their preparation typically involves multistep processes and organic solvents, which may complicate large-scale production and limit encapsulation efficiency for chemically heterogeneous extracts. In addition, their degradation behavior and release kinetics in the digestive environment may be difficult to predict [11,12]. Conventional SLNs have also been investigated for natural product delivery; however, their highly ordered lipid matrices often restrict drug accommodation and may promote compound expulsion during lipid crystallization, particularly when accommodating complex lipophilic mixtures [13].
In this context, nanostructured lipid carriers (NLCs) have emerged as versatile colloidal systems for the incorporation of poorly water-soluble compounds and complex natural extracts [14]. By combining solid and liquid lipid components, NLCs form partially disordered matrices capable of accommodating high payloads while maintaining colloidal stability [15]. For chemically heterogeneous natural extracts such as propolis, where multiple constituents with different polarity and partitioning behavior coexist, this structural characteristic is particularly advantageous because it enables the incorporation of diverse lipophilic constituents while minimizing crystallization-induced expulsion phenomena [16]. Importantly, NLCs should not be regarded solely as passive encapsulation vehicles. Their internal organization, interfacial properties, and interactions with surrounding media govern not only loading efficiency and physical stability but also the fraction of encapsulated compounds that become accessible under specific physicochemical environments [17]. In this sense, lipid-based nanocarriers can actively modulate compound partitioning, apparent release, and analytical accessibility, particularly in complex biorelevant media.
For chemically heterogeneous systems such as propolis, rational extract selection represents a critical initial step. Antioxidant profiling, when interpreted as a physicochemical descriptor rather than a direct predictor of biological efficacy, provides a comparative chemical framework to guide this selection. The use of complementary antioxidant assays offers an integrated view of redox-related properties and reduces the risk of advancing extracts whose apparent activity is dominated by assay-specific effects or narrow subsets of reactive constituents [18]. This strategy is especially relevant when the objective is to investigate how a lipid-based nanosystem modulates accessibility and release behavior, rather than to rank extracts solely on the basis of bioactivity claims.
In parallel, the gastrointestinal environment introduces additional complexity for lipid-based nanocarriers. Bile salts and phospholipids are surface-active amphiphiles capable of inducing interfacial remodeling, mixed colloid formation, and redistribution of lipophilic compounds [19]. Importantly, structural alteration of lipid carriers in bile-containing media does not necessarily translate into proportional increases in analytically measured release, as morphological changes and assay-accessible fractions represent distinct physicochemical readouts. Understanding this distinction is essential for the rational interpretation of release data obtained in biorelevant media.
Unlike most previous studies on propolis delivery systems, which primarily focus on improving encapsulation efficiency or biological activity, the present work investigates how a lipid nanosystem modulates the physicochemical accessibility and apparent release of a chemically complex extract under biorelevant gastrointestinal conditions. Specifically, this study aimed to develop and characterize a Gelucire®-based nanostructured lipid carrier loaded with a selected Chilean propolis extract from the Metropolitan Region and to investigate how simulated gastrointestinal environments containing bile components influence carrier structure and apparent propolis release. We hypothesized that the nanostructured lipid carrier would behave as a retentive lipid matrix under simple aqueous buffer conditions, while bile salts and phospholipids present in biorelevant media would induce composition- and pH-dependent interfacial remodeling and redistribution of propolis into mixed colloidal assemblies. Such processes were expected to alter the assay-accessible fraction without necessarily implying complete carrier disintegration. To address these questions, propolis extracts from different local origins were first screened using complementary antioxidant assays to support rational extract selection. The selected extract was subsequently incorporated into NLCs, followed by systematic evaluation of loading efficiency, colloidal properties, and physical stability. Release behavior was assessed in aqueous buffers and bile-component-containing simulated gastrointestinal media, alongside morphological analysis by transmission electron microscopy. Finally, a preliminary cytocompatibility assessment in HepG2 cells was conducted to support the potential relevance of the developed nanosystem for future biomedical applications.
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Materials
Gelucire® 44/14 was donated by Gattefossé (Saint-Priest, France). Miglyol® 812, Tween® 80, dimethyl sulfoxide (DMSO, Molecular Biology grade), Folin–Ciocalteu reagent, sodium carbonate (Na2CO3), gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH•, ≥90%), ABTS Single Reagent, fluorescein, 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH, 97%), sodium chloride (NaCl), L-α-lecithin (lecithin), pepsin, hydrochloric acid (HCl), sodium taurocholate, sodium hydroxide, acetic acid, sodium phosphate dibasic (Na2HPO4), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT, ≥97.5%), and Amicon® Ultra 10 kDa were purchased from Merck (Merck KGaA, Darmstadt, Germany). SnakeSkin ™ dialysis tubing (cellulose membrane, 10 kDa MWCO) was purchased from Thermo Fisher Scientific (Carlsbad, CA, USA). Chilean crude propolis samples were obtained from three locations in the Metropolitan Region of Chile: Peñaflor (33°37′00″ S, 70°55′00″ W), Pudahuel (33°26′42″33 S, 70°44′28″7 W), and Pirque (33°37′48″ S, 70°34′12″ W). Dulbecco’s Modified Eagle Medium (DMEM high-glucose; Cat# SH30243.02), phosphate-buffered saline (PBS, without calcium and magnesium; Cat# SH302560.1), fetal bovine serum (FBS; Cat# SV30160.03), and penicillin/streptomycin (Cat# SH40003.01) solution were obtained from Cytiva (Marlborough, MA, USA). Trypsin-EDTA solution (0.25%; Cat# 25200-072) was purchased from Thermo Fischer Scientific (Gibco, Waltham, MA, USA). Ultra-pure water (18.2 MΩ·cm) was produced using a Simplicity System from Millipore (Merck KGaA, Darmstadt, Germany). All materials were used as received without any further purification.
Carrasco-Rojas, J.; Solas-Soto, J.; Veas-Albornoz, R.; Lagos, C.F.; Simirgiotis, M.J.; Arriagada, F.; Ortiz, A.C. Nanostructured Lipid Carriers as Physicochemical Modulators of Complex Natural Extracts: Release Behavior and Bile-Induced Remodeling in Biorelevant Media. Molecules 2026, 31, 1028. https://doi.org/10.3390/molecules31061028
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