A Comparative Study of the Influence of Lipid Composition on Stability, In Vitro Release, and Antioxidant Activity of Quercetin-loaded Ceramide-containing Liposomes for Topical Delivery

Abstract

This study investigates the influence of phospholipid saturation by comparing hydrogenated soy phosphatidylcholine (HSPC) and egg yolk phosphatidylcholine (EPC) on the physicochemical characteristics, colloidal stability, drug release behavior and antioxidant activity of quercetin-loaded ceramide-containing liposomes for topical delivery. Liposomes composed of EPC:Cer:Que and HSPC:Cer:Que were prepared by thin-film hydration followed by sonication. The nanosystems were studied for particle size, polydispersity index, ζ-potential, and entrapment efficiency. Colloidal stability was evaluated under mechanical stress, accelerated aging, and long-term storage, while in vitro drug release, drug retention, and antioxidant activity were assessed under simulated skin conditions. Incorporation of ceramides into EPC bilayer reduced stability issues associated with unsaturated phospholipids and maintained a fluid structure, promoting drug release. Both formulations exhibited enhanced colloidal stability with EPC-based liposomes maintaining their properties at all conditions, whereas HSPC-based liposomes showed increased particle size following mechanical stress. HSPC-based liposomes demonstrated higher quercetin entrapment efficiency (63 ± 5%), improved retention over time (75% at 90 days), and a more sustained release (45% at 480 min). EPC-based ceramide-containing liposomes exhibited faster release (50% at 240 min), resulting in greater antioxidant activity as indicated by DPPH assay (0.474 ascorbic acid equivalents), while FRAP assay results were comparable for both formulations (0.012 Fe²⁺ equivalents), indicating consistent ferric reducing potential after release. These findings highlight the significance of phospholipid composition in liposome behavior and provide insights into the design of stable and effective ceramide-containing nanosystems for topical delivery of poorly water-soluble compounds such as quercetin, with potential applications in managing photoaging, inflammation, and wound healing.

Introduction

Quercetin is a natural flavonoid biomolecule, which has gained significant attention, exhibiting anti-inflammatory, antioxidant, antimicrobial, and anti-cancer properties [1,2,3,4]. However, the lipophilic nature, low oral absorption, and enzymatic degradation of quercetin limits its clinical application. To address these challenges, several nanosystems have been developed, mainly lipid-based, for enhancing quercetin delivery and bioavailability [5,6,7,8].

Among a wide range of nanocarriers, liposomes exhibit several advantages for quercetin delivery. Liposomes are colloidal, pseudo-spherical vesicles composed of lipid bilayers and an aqueous core [9]. These vesicles are formed as amphiphilic molecules, such as phospholipids, self-assemble in an aqueous environment. The unique structure of liposomes allows the entrapment of both hydrophilic and lipophilic molecules, making them suitable nanocarriers in various pharmaceutical and biomedical applications [10, 11]. Moreover, liposomes are widely utilized in drug delivery due to their biocompatible and biodegradable nature, as well as their ability to protect drugs from degradation, and potential for targeted delivery through surface modifications [12, 13].

Lipid composition plays a crucial role in liposome properties including particle size, membrane rigidity, fluidity, colloidal stability, and surface charge [12, 14, 15]. Specifically, saturated phospholipids, such as hydrogenenated soybean phosphatidylcholine (HSPC) and dipalmitoylphospharidylcholine (DPPC), enhance membrane stability and drug retention as they form more rigid lipid bilayers with limited permeability [16, 17]. In contrast, unsaturated phospholipids, such as egg yolk phosphatidylcholine (EPC) increase membrane fluidity, promoting more efficient drug release, whereas lead to reduced structural stability [18, 19].

Beyond systemic administration, liposomes have been used as nanocarriers for topical drug delivery, especially for enhancing the permeability and efficacy of bioactive molecules through the skin [20,21,22,23]. Due to their similarity to biological membranes, liposomes facilitate drug penetration, while also offering controlled release and enhanced stability. These properties make them suitable nanocarriers for increasing the biodistribution of biomolecules, reducing side effects, and improving the skin absorption of both hydrophilic and lipophilic compounds [24,25,26,27].

In this context, ceramides are incorporated into liposomal nanosystems for topical application, due to their crucial role in skin barrier function, hydration, and structural integrity. As major components of the stratum corneum (SC), they regulate cell adhesion, epidermal differentiation, and prevent transepidermal water loss [28,29,30]. At least 21 distinct ceramide subclasses have been identified in the SC, with variations in headgroup and fatty acid chain length [31]. However, their application is limited due to their hydrophobic nature. To overcome this challenge, ceramides are incorporated into liposomes to enhance the stability, bioavailability, and skin penetration of biomolecules. Several ceramide-containing liposomes have been developed to improve skin permeation of bioactive molecules and provide controlled drug release [18, 32, 33]. When ceramides are combined with unsaturated phospholipids, such as egg yolk phosphatidylcholine (EPC), the resulting lipid bilayers exhibit increased fluidity and permeability [18]. This leads to more efficient drug release and may compromise structural stability issues of pure EPC lipid bilayers. Ceramide-based liposomes can also promote fusion with the stratum corneum, thus improving drug delivery. Although various lipid-based formulations have been investigated for topical quercetin delivery, there is limited understanding of how the saturation level of phospholipids influences the structural and functional properties of ceramide-containing liposomes.

The objective of this research was to develop ceramide-containing liposomal nanosystems for topical quercetin delivery and to evaluate the impact of lipid composition on their stability, entrapment efficiency, and release kinetics under simulated skin conditions. Specifically, ceramides, essential components of the skin barrier, were incorporated into liposomes composed of either hydrogenated soy phosphatidylcholine (HSPC) or egg yolk phosphatidylcholine (EPC) to enhance quercetin skin penetration. HSPC exhibits greater membrane rigidity and enhanced stability due to its high saturation, whereas EPC provides a more flexible membrane structure that can improve skin permeation and drug release. This study provides insights into the potential of HSPC:Ceramides:Quercetin (2:1:0.5 molar ratio), and EPC:Ceramides:Quercetin (2:1:0.5 molar ratio) nanosystems for topical quercetin delivery. To the best of our knowledge, this is the first report to compare the impact of phospholipid saturation, specifically hydrogenated soy phosphatidylcholine (HSPC) and egg yolk phosphatidylcholine (EPC), on the structural and functional integrity of ceramide-containing liposomes for topical quercetin delivery.

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Materials

The liposomal nanosystems were prepared using L-α-phosphatidylcholine, hydrogenated (Soy) and L-α-phosphatidylcholine (95%) (Egg, Chicken), both obtained from Avanti Polar Lipids Inc. (Alabaster, AL, USA). The ceramide mixture derived from a natural bovine source was purchased from Matreya LLC (State College, PA, USA). Quercetin was obtained from Fluka BioChemika (Buchs, Switzerland). Ferrous chloride hexahydrate was obtained by Panreac Applichem (Barcelona, Spain), 2,4,6-tripyridyl-s-triazine (TPTZ) by Alfa Aesar (Stoughton, MA, USA), and ferrous sulfate heptahydrate by Riedel de Haen (Seelze, Germany). Chloroform, methanol, ethanol, and H2O (all HPLC grade) were purchased from Fisher Scientific (UK). 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ascorbic acid were obtained from Sigma-Aldrich (Darmstadt, Germany).

Tsichlis, I., Koufonikola, VD., Chaikali, C. et al. A Comparative Study of the Influence of Lipid Composition on Stability, In Vitro Release, and Antioxidant Activity of Quercetin-loaded Ceramide-containing Liposomes for Topical Delivery. AAPS PharmSciTech 26, 235 (2025). https://doi.org/10.1208/s12249-025-03226-0

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