Development and Physicochemical Characterization of Trans-Resveratrol Liposomes: Influence of Phospholipid Composition and Ultrasound-Assisted Processing

Background/Objectives: Trans-resveratrol is a bioactive polyphenol with limited pharmaceutical applicability due to poor aqueous solubility, chemical instability, and low bioavailability. This study aimed to develop trans-resveratrol-containing liposomal formulations and evaluate the influence of phospholipid composition and ultrasound-assisted processing (UAP) using a conventional ultrasonic bath on their physicochemical properties and storage behavior.

Methods: Liposomes were prepared by the ethanol injection method using Lipoid^® S75, Lipoid^® P45, soybean lecithin, egg yolk lecithin, and selected binary phospholipid mixtures. Trans-resveratrol identity was verified by LC–MS. Particle size, polydispersity index (PDI), and zeta potential were determined before and after UAP, and physical stability was assessed after 30 days of refrigerated, light-protected storage.

Results: Phospholipid composition markedly influenced liposome physicochemical characteristics. Lipoid^® P45-based formulations exhibited favorable initial particle size, PDI, and zeta potential profiles. UAP generally reduced particle size and improved particle size distribution immediately after preparation, although the magnitude of the effect depended on formulation composition. During refrigerated storage, short-term physical storage behavior was strongly formulation-dependent. Selected trans-resveratrol-containing formulations, particularly Lr12, Lr13, and Lr15, exhibited marked particle growth and increased polydispersity, whereas Lr20–Lr22 exhibited the most favorable particle size and PDI profiles after 30 days.

Conclusions: Phospholipid composition was a critical determinant of trans-resveratrol liposome performance. Conventional ultrasonic-bath processing provided a simple and accessible means of improving initial liposomal characteristics, but did not universally ensure short-term physical stability during 30 days of refrigerated storage. Selected binary phospholipid formulations, particularly Lr20–Lr22, showed favorable physicochemical profiles and may represent promising candidates for further formulation development.

Introduction

Liposomes are among the most extensively investigated nanocarrier systems for the delivery of bioactive compounds with limited aqueous solubility and poor physicochemical stability. Their phospholipid bilayer structure enables the encapsulation of both hydrophilic and lipophilic molecules, providing protection against environmental degradation, improving aqueous dispersion, and potentially enhancing bioavailability [1,2]. Owing to their excellent biocompatibility, biodegradability, and versatility, liposomes have been widely explored for pharmaceutical, nutraceutical, and cosmetic applications [3,4,5].

Trans-resveratrol (3,5,4′-trihydroxystilbene) is a naturally occurring polyphenolic compound predominantly found in grapes, berries, peanuts, and red wine [6,7]. It has attracted considerable attention because of its antioxidant, anti-inflammatory, cardioprotective, neuroprotective, antimicrobial, and anticancer activities [8,9,10,11,12]. Despite these promising pharmacological properties, the clinical and pharmaceutical application of trans-resveratrol remains limited by its poor aqueous solubility, susceptibility to photoisomerization and oxidation, rapid metabolism, and consequently low oral bioavailability [12,13]. These limitations substantially reduce its therapeutic potential and have stimulated intensive research into alternative delivery systems capable of improving its stability and bioavailability [14].

Among the various nanocarrier platforms investigated for trans-resveratrol delivery, liposomes represent one of the most promising approaches because the lipophilic structure of trans-resveratrol allows its incorporation into the phospholipid bilayer, thereby protecting the compound from degradation while improving its dispersion in aqueous media. Previous studies have demonstrated that liposomal encapsulation may enhance the physicochemical stability, controlled release, and biological activity of trans-resveratrol [15,16,17,18]. However, the characteristics of liposomal formulations are highly dependent on formulation variables, including phospholipid composition, preparation method, and post-processing conditions.

Phospholipid composition plays a fundamental role in liposome formation and stability. Differences in phosphatidylcholine content, fatty acid composition, and lipid purity influence membrane fluidity, vesicle formation, particle size, surface charge, and long-term stability [19]. Commercial phospholipids such as Lipoid® S75, Lipoid® P45, soybean lecithin, and egg yolk lecithin differ considerably in their composition and may therefore produce liposomes with distinct physicochemical characteristics. Although these phospholipids have been used individually in liposomal formulations, comparative studies evaluating their performance under identical formulation conditions remain limited.

Besides lipid composition, post-formulation processing is another important factor affecting liposome quality. Various size-reduction techniques, including extrusion, high-pressure homogenization, and probe ultrasound homogenization, have been employed to obtain nanosized liposomes with narrow particle size distributions [20]. However, these techniques frequently require specialized equipment and relatively high operating costs. In contrast, ultrasound-assisted processing using a conventional ultrasonic bath represents a simple, inexpensive, and widely accessible alternative that may improve liposomal dispersions without the need for sophisticated instrumentation. Despite its routine use in laboratory practice, its influence on the physicochemical characteristics of trans-resveratrol liposomes has received comparatively little attention.

Particle size, PDI, and zeta potential are considered critical quality attributes of liposomal formulations because they directly influence colloidal stability, reproducibility, storage behavior, and potential biological performance [21,22]. Therefore, systematic evaluation of these parameters is essential for selecting suitable phospholipid systems and assessing the effectiveness of post-formulation processing.

The present study aimed to develop trans-resveratrol-containing liposomal formulations using different natural phospholipid compositions prepared by the ethanol injection method and to evaluate the influence of ultrasound-assisted processing using a conventional ultrasonic bath on their physicochemical characteristics and short-term physical storage behavior. In addition, control liposomal formulations without trans-resveratrol were included to distinguish the effects of phospholipid composition from those associated with the presence of trans-resveratrol in the formulation. The findings provide practical guidance for the development of simple and reproducible liposomal formulations using an accessible post-processing approach.

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Materials

Trans-resveratrol (≥98% purity) was used as the model lipophilic bioactive compound in all formulations. Prior to liposome preparation, its identity was verified by liquid chromatography–mass spectrometry (LC–MS).

Four natural phospholipid materials with different phosphatidylcholine contents were selected for liposome preparation to evaluate the influence of lipid composition on the physicochemical characteristics of the formulations. Lipoid^® S75 (≥70% phosphatidylcholine) and Lipoid^® P45 (≥45% phosphatidylcholine) were obtained from Lipoid GmbH (Ludwigshafen, Germany). Soybean lecithin and egg yolk lecithin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethanol (96%, pharmaceutical grade) was obtained from Stumbras (Kaunas, Lithuania), while purified water was produced using a Milli-Q water purification system (Millipore, Bedford, MA, USA).

The physicochemical properties of the liposomal formulations were determined using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK), which employs dynamic light scattering (DLS) for particle size and PDI measurements and electrophoretic light scattering (ELS) for zeta potential determination. Ultrasound bath-assisted processing was performed using a SONOREX DIGITEC DT 156 BH ultrasonic bath (BANDELIN electronic GmbH & Co. KG, Berlin, Germany), and lipid dispersions were prepared using an IKA C-MAG HS7 magnetic stirrer (IKA-Werke GmbH & Co. KG, Staufen, Germany) and a Heidolph Laborota 4000 efficient rotary evaporator (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). Lipid solutions were introduced into the aqueous phase using a Landgraf LA-120 syringe pump (Landgraf Laborsysteme HLL GmbH, Langenhagen, Germany). Sample preparation was performed using calibrated analytical balances (Shimadzu AUW120D, Kyoto, Japan) and adjustable micropipettes (Eppendorf AG, Hamburg, Germany).

All chemicals and reagents were of analytical or pharmaceutical grade and were used as received without further purification.

Pudžiuvelytė, L.; Paliulian, A. Development and Physicochemical Characterization of Trans-Resveratrol Liposomes: Influence of Phospholipid Composition and Ultrasound-Assisted Processing. Pharmaceutics 2026, 18, 1303. https://doi.org/10.3390/pharmaceutics18101303